METHODS AND COMPOSITIONS FOR THE TREATMENT OF CANCER WITH 5-IODO-2-PYRIMIDINONE-2(prime)-DEOXYRIBOSE (IPdR)

ABSTRACT

Provided are methods of treating a human patient having cancer by administering IPdR to the patient in the form of an oral drug and administering radiation therapy (RT) to the patient. The method can also include administering a chemotherapeutic drug or biologic agent to the patient. Also provided are methods for optimizing IPdR sensitization for radiation therapy for a cancer patient having been administered IPdR.

REFERENCE TO RELATED APPLICATIONS

This continuation application claims priority to and the benefit of U.S.Provisional Application No. 62/060,688, filed Oct. 7, 2014, and PCTApplication No. PCT/US2015/054534 filed Oct. 7, 2015, the entirecontents of each of which are hereby incorporated by reference herein.

REFERENCE TO FEDERAL FUNDING

This invention was made with government support under: RO1 CA 50595awarded by the National Institutes of Health and the National CancerInstitute; R44 CA 76835 awarded by the National Institutes of Health;RAID #197 awarded by the National Cancer Institute; andHHSN261201400013C awarded by the National Cancer Institute. Thegovernment has certain rights in the invention.

FIELD OF THE INVENTION

The invention relates to methods and compositions for treating cancer.In particular, the invention relates to methods and compositions fortreating cancer by administering 5-iodo-2-pyrimidinone-2′-deoxyribose(IPdR) to a patient in need thereof.

BACKGROUND

In 2015, it was estimated that 1,658,370 men and women in the U.S. wouldbe diagnosed with cancer, and nearly 590,000 cancer patients would dieof their disease. Currently, cancer is the second leading cause of deathin the U.S. and is projected to surpass the death rate from heartdisease in the next few years.

Radiation therapy (RT) plays a critical role in the treatment of mostcommon human cancers, with over 50% of all cancer patients receiving RTat some point during the course of their treatment. It is estimated thatover 40% of cured cancer patients have received RT as part of theircancer treatment(http://www.rtanswers.org/statistics/aboutradiationtherapy).Consequently, any invention that improves the efficacy and/or reducesthe toxicity profile of RT will have a profound impact on cancertreatment in the U.S. and worldwide.

RT kills cells via several biological and molecular mechanisms, some ofwhich are well understood, while others need further elucidation. RTdoes not kill only cancer cells, but can also kill normal cells withinthe RT treatment volume in a patient, leading to both acute (occurringduring or immediately following RT) and late (occurring months to yearsfollowing RT) normal tissue toxicities. In principle, all cancers can becontrolled if sufficiently high radiation doses can be delivered totumor cells; however, in practice, the radiation dose is frequentlylimited by toxicities resulting from exposure of normal tissues to highradiation doses. The balance between the probability of tumor controland the risk of normal tissue complications is a measure of thetherapeutic index (TI). Normal tissue damage cannot be completelyavoided because the radiation doses necessary to achieve tumor controltypically overlap with those that can cause acute and/or late normaltissue toxicities. The goal, therefore, is to develop strategies thatcan selectively increase the radiation effect on the tumor while sparingthe normal cells and tissues.

A major RT strategy employed to achieve this goal is to target definedvolumes of cancerous tissue while attempting to minimize RT exposure toadjacent normal tissues. Over the last 3 decades, there have beensignificant improvements in our technical capabilities to better targettumor tissues and limit RT exposure to normal tissues by utilizingtechnologies such as 3-dimensional conformal RT, intensity-modulated RT,image-guided RT as well as the more costly proton beam RT and heavy ionRT. However, significant RT dose limiting acute and late normal tissuetoxicities are observed in clinical testing of these new technologieswith the use of modest RT dose escalations. Indeed, two recent reviewsconclude that the tumor control probability of these new RT technologiesfor common cancers such as rectal cancer, high-grade gliomas, and headand neck cancer will be increased by <10% at best.

So, target refinement as a strategy to improve the efficacy and/ortoxicity of RT has led to only limited improvements.

Another strategy has been to use systemic cytotoxic chemotherapy inconjunction with RT in an attempt to further improve tumor control. Inaddition to the direct killing of the cancer cells by the drug, some ofthese agents have been observed to enhance the killing effects of RTwhen the two modalities are administered together. Currently, cytotoxicchemotherapy drugs such as 5-fluorouracil (5-FU), capecitabine (a 5-FUprodrug), cisplatinum, carboplatinum, and to a lesser extent,mitomycin-C, gemcitabine, irinotecan, taxol, oxaliplatinum andtemozolomide are used as potential clinical radiosensitizers for a largenumber of common cancers including rectal cancer (42,000 new cases),lung cancer (228,000 new cases), head and neck cancers (41,000 newcases), pancreas cancer (45,000 new cases), anal cancer (7,000 newcases) and high grade brain cancers (14,000 new cases), based on 2015cancer incidence data. Less commonly, biologics such as the humanizedmonoclonal antibodies cetuximab (targeting the epidermal growth factorreceptor) and bevacizumab (targeting the vascular endothelial growthfactor receptor) are used during RT either alone or in combination withsome of the above cytotoxic chemotherapy drugs.

However, none of these drugs or biologics were developed as specificclinical radiosensitizers and all have significant single agent acuteand late normal tissue toxicities, which can be further enhanced whenused during RT. It is now clear that most currently used concomitantchemo/biologic-RT combinations are delivered close to (or at) the limitsof normal tissue tolerances such that further intensification byincreasing the cytotoxic drug dose or by adding different classes ofcytotoxics or biologics appears not to be a viable treatment strategyfor these common cancers.

A third strategy to improve the efficacy/toxicity profile of RT is touse a drug(s) that specifically targets mechanisms of tumor cellresistance to RT, thereby making the tumor more susceptible to thedamaging effects of RT relative to the normal tissues. While it is anappealing strategy, there are currently no drugs with a specific FederalDrug Administration (FDA) or European Medicines Agency (EMA) approvedindication of radiosensitization. However, it is well recognized thatthe primary cellular target of RT is DNA. RT kills cells by causingirreversible DNA strand breaks, making them unable to divide andproliferate. Well over 50 years ago, it was recognized that certaindrugs, called halogenated nucleoside analogs, are falsely incorporatedinto DNA, and when these cells with the defective DNA are exposed to RT,tumor cell killing is increased by up to two-three fold compared tocells without the defective DNA. To date, the nucleoside analog that hasbeen found to be the most effective as a specific radiosensitizing drugis 5-iodo-2′-deoxyuridine (IUdR). Clinical trials during the1990's-early 2000's showed that IUdR enhanced the effectiveness of RT inthe treatment of RT-resistant brain and soft tissue/bone cancers(summarized in Tables 1 and 2). However, the use of IUdR is completelyimpractical; that is, it needs to be administered as a continuousintravenous infusion, 24 hours/day for 5-6 weeks during RT. ProlongedIUdR infusions also caused systemic normal tissue toxicities, especiallymyelosuppression and GI toxicities. Consequently, Phase III clinicaltrials were not performed and IUdR is not approved for clinical use.

More recently, a newer nucleoside analog,5-iodo-2-pyrimidinone-2′-deoxyribose (IPdR) has been developed. IPdR istaken by mouth (po) and is a prodrug of IUdR, that is, when IPdR isingested, the body converts it into IUdR, so IPdR is essentially a wayto conveniently administer a drug, IUdR, that is known to enhance theeffectiveness of RT.

IPdR has thus been specifically developed as a radiosensitizing drug;this development having been sponsored by the National Cancer Institute(NCI) through a series of extramural and intramural grants over twodecades. The rigid guidelines for the preclinical and early phaseclinical assessment of novel radiosensitizers like IPdR have beenpublished by the Radiation Modifier Working Group of the U.S. NCI, aswell as by the UK National Cancer Research Institute (NCRI). Theextensive preclinical studies of IPdR passed the rigid guidelines fromboth the U.S. NCI and UK NCRI Working Groups, as well as the U.S. FDA,leading to the filing of an Investigational New Drug (IND) submission.Importantly, po IPdR was found to be more effective as a tumorradiosensitizing drug in animals with little normal tissue toxicitieswhen directly compared to continuous intravenous infusions of IUdR. Mostrecently (April, 2013), intramural NCI investigators published thefirst-in-human Phase 0 trial of single doses of po IPdR in patients withadvanced malignancies. This study concluded that adequate plasma levelsof the active drug, IUdR, from the oral prodrug, IPdR, were achieved,justifying proceeding with a Phase I trial of IPdR in combination withradiation. Indeed, the U.S. NCI is currently sponsoring two Phase I andPharmacokinetic (PK) trials combining escalating doses of IPdR givenorally, once daily×28 days during RT for patients with metastaticgastrointestinal cancers, and for patients with brain metastases.

SUMMARY OF THE INVENTION

The present invention relates, in part, to the use of IPdR as aradiosensitizing drug for treating cancer patients. The presentinvention also relates, in part, to assays that correlate IUdR-DNAcellular incorporation in tumors and normal tissues to improve thetherapeutic index (TI) for IPdR and RT combination therapy and toidentify groups of patients that may benefit from the use of IPdR andRT.

The present invention generally relates to the use of5-iodo-2-pyrimidinone-2′-deoxyribose (IPdR), an oral (po) prodrug of5-iodo-2′-deoxyuridine (IUdR), used during radiation therapy (RT) tomaximally enhance IUdR-mediated tumor radiosensitization whileminimizing normal tissue toxicities, resulting in an improvedtherapeutic index (TI). In various embodiments, the methods andcompositions of the invention are used to treat “difficult to cure”human solid cancers, including cancers with genetic/epigeneticdeficiencies in DNA mismatch repair (MMR) and/or base excision repair(BER). This invention also relates to the clinical development of poIPdR used during RT+specific chemotherapy combinations, particularlyfluoropyrimidines, platinum analogs, temozolomide, and ribonucleotidereductase inhibitors, to maximally enhance IUdR-mediated tumor chemo-and radio-sensitization resulting in an improved TI for the combinedmodality treatment in solid cancers where use of those chemotherapeuticagents are indicated.

Additionally, the invention involves the development of assays that willallow assessment of the radiosensitization effect of IPdR, andspecifically, the use of those assays to predict disease response inindividuals or groups of patients. In some embodiments, these assaysinvolve cellular measurements of % IUdR-DNA incorporation in tumor cellsand in potential IUdR-dose-limiting normal tissue cells that, whenmeasured in combination, may provide an intermediate prediction of TI(i.e. efficacy and toxicity) during po IPdR+RT treatment.

In one aspect, the invention provides methods of treating a humanpatient having cancer. The method includes the steps of administeringIPdR to the patient in the form of an oral drug; and administeringradiation therapy (RT) to the patient. These methods are particularlyuseful if a patient cannot tolerate the optimum, or standard of care,levels of radiation treatment without the addition of IPdR. Thesemethods also are particularly suitable for treating sporadicMMR-deficient cancers.

In another aspect, the invention provides methods of treating a humanpatient having cancer that include the steps of administering IPdR tothe patient in the form of an oral drug; administering achemotherapeutic drug or biologic agent to the patient; andadministering radiation therapy (RT) to the patient. These methods areparticularly suitable for treating BER-deficient cancers.

In another aspect, the invention provides methods for optimizing IPdRsensitization for radiation therapy for a cancer patient having beenadministered IPdR. These methods include the steps of determining anIUdR-DNA incorporation level in a first tumor biopsy taken from thepatient; determining an IUdR-DNA incorporation level in a normal tissuesample; and calculating a therapeutic index; wherein the therapeuticindex guides dose and schedule of radiation therapy. In variousembodiments, the therapeutic index is calculated by dividing the percentIUdR-DNA incorporation level in the first tumor biopsy by the percentIUdR-DNA incorporation of the normal tissue sample. In variousembodiments, the tumor biopsy and normal tissue sample are obtained fromthe same patient, and in some embodiments the samples are obtained priorto administering radiation therapy. In various embodiments, IUdR-DNAincorporation levels are measured by high performance liquidchromatography (HPLC) or flow cytometry (e.g., using anti-IUdRantibodies). In various embodiments, the method includes the step ofdetermining an IUdR-DNA incorporation level in a second tumor biopsytaken at a second time from the patient, determining an IUdR-DNAincorporation level in a normal tissue sample; and calculating atherapeutic index. In various embodiments, the normal tissue sampleincludes circulating cells (e.g., granulocytes) or oral mucosa. Thetumor biopsies and tissue samples can be obtained at periodic intervals,such as weekly or monthly intervals.

Embodiments of any of the foregoing aspects can include one or more ofthe following steps or features:

In various embodiments, radiation therapy is administered in atherapeutically effective amount. Radiation therapy can be deliveredusing a hyperfractionated technique. In addition, the radiation therapycan be delivered using one or more of the following techniques:3-dimensional conformal radiation therapy, intensity-modulated radiationtherapy, image-guided radiation therapy, tomotherapy, stereotacticradiosurgery and stereotactic body radiation therapy. The source ofradiation therapy can be any suitable source, such as, for example,protons and carbon ions.

In various embodiments, a therapeutically effective amount of achemotherapeutic drug or biologic agent is administered or provided tothe patient. The chemotherapeutic drug can be a fluoropyrimidine, suchas, by way of non-limiting example, 5-fluorouracil, floxuridine,capecitabine, DPD-inhibiting fluoropyrimdines, and combinations thereof.The chemotherapeutic drug can be a platinum analog such as, by way ofnon-limiting example, cisplatinum, carboplatinum, oxaliplatinum, andcombinations thereof. The chemotherapeutic drug can be a ribonucleotidereductase inhibitor, such as, by way of non-limiting example,hydroxyurea, gemcitabine, triapine, COH29, and combinations thereof. Thechemotherapeutic drug also can be a methylating agent or a MMRmodulator. The biologic agent can be, for example, a BER modulator.

In various embodiments, the cancer or tumor is a solid tumor.

In various embodiments, IPdR is administered at a dose of 0.1-50gm/M2/day, and more preferably between 2-5 gm/M2/day. For example, IPdRcan be administered at a dose of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6,0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 6, 7, 8, 9,10, 15, 20, 25, 30, 35, 40, 45, or 50 gm/M2/day, and any range or valuebetween about 0.1-50 gm/M2/day.

DETAILED DESCRIPTION

To facilitate understanding of the invention, a number of terms aredefined below.

A radiosensitizer (or radiation sensitizer) is a drug or compound thatis capable of being incorporated into cellular DNA and subsequentlyenhances the DNA damage caused by ionizing radiation (IR) when humansolid cancers are treated with radiation therapy (RT).5-iodo-2′-deoxyuridine (IUdR) is an analog of thymidine (TdR) that isreadily taken up by cells via active nucleoside transport and isinitially phosphorylated to the monophosphate derivative (IdUMP) by therate-limiting enzyme, thymidine kinase (TK). IdUMP is sequentiallyphosphorylated intracellularly to the triphosphate derivative, IdUTP,which is then used in DNA replication, in direct competition withdeoxythymidine triphosphate (dTTP), by DNA polymerase. TdR replacementby IUdR results from stereochemical similarities between the Van derWaal's radius of the iodine atom (2.15 Angstrom) and the methyl group(2.0 Angstrom) at the 5-position of the uridine molecule. IUdR is notefficiently absorbed when administered orally; therefore, requiringintravenous (IV) or intra-arterial (IA) administration. Unfortunately,the plasma half-life of IV or IA IUdR is <5 minutes, with rapidcatabolism of the drug, principally by the liver, to the free base,followed by dehalogenation. Consequently, prolonged infusions of IUdRare necessary for it to be effective as a clinical radiosensitizer.

A prodrug is a compound that, upon administration, must undergo chemicalconversion by metabolic processes before becoming an activepharmacological agent.

Prodrugs are often designed to improve bioavailability when the activedrug itself is poorly absorbed from the gastrointestinal tract. Withrespect to the present invention, 5-iodo-2-pyrimidinone-2′-deoxyribose(IPdR) is a prodrug of IUdR with excellent bioavailability whenadministered orally in non-human species (mice, rats, ferrets andmonkeys) as well as in humans. An aldehyde oxidase, principally inliver, efficiently converts IPdR to IUdR.

Similarly, the active drug, IUdR or it's oral prodrug, IPdR, may alsoenhance the cytotoxicity of several different clinically relevant cancerchemotherapy drugs, and as such, can be classified as a chemosensitizer(or chemotherapy modifying drug). The interaction of IUdR (or IPdR) withthese cancer chemotherapy drugs can be quantified experimentally usingbiostatistical methodologies as defined below. Such chemotherapydrug+IR+IPdR combinations are disclosed herein.

The evaluation of the present invention in pre-clinical studies of IPdRas an in vivo radiosensitizer and IUdR as an in vitro radiosensitizerand chemosensitizer required biostatistical tests of interaction, i.e.less than additive (antagonistic), additive, or greater than additive(synergistic) effects of IR±IUdR (or IPdR) and chemotherapydrug/biologic±IUdR (or IPdR). For the in vitro studies using clonogenicsurvival data of human cancer cells, the response of interest is not asingle value, but rather a dose-response relationship or curve (oversome defined range of doses). A radiation cell survival curve is adose-response relationship between the ionizing radiation (IR) doseapplied to cells and the proportion of cells surviving the IR exposure.A chemotherapy cell survival curve is a similar relationship between thedose of a chemotherapy agent (drug/biologic) and cell survival.

Alternatively, other biostatistical methodologies can be used inexperimental in vitro (cellular) or in vivo (animals; typically, athymicmice with human cancer tumor xenografts) assays to measure synergistic(greater than additive) effects of IUdR or IPdR and IR or modifiers ofIR-induced DNA damage repair. Additionally, computational in silicomodels have been developed and used to quantitatively analyze theeffects of DNA Mismatch Repair (MMR) and Base Excision Repair (BER)processing of IUdR-DNA cellular incorporation on the effectiveness ofthe pre-clinical treatment strategies where IUdR (or IPdR) and/or IR areused. These in silico models of the two different DNA repair processes(i.e. MMR and BER) have been developed at both the cellular level andthe molecular level. The MMR status and the BER status of both normaland human cancer cells/tissues are important for determining the %IUdR-DNA cellular incorporation, the cell cycle response to differentlevels of % IUdR-DNA cellular incorporation, as well as the cytotoxic(cell death) response to IPdR+IR. The results of these extensivepre-clinical in vitro, in vivo, and in silico modelings have served asimportant milestones leading to the current human clinical developmentof IPdR as disclosed herein.

Pharmacokinetic (PK) studies are intended to define the time course of adrug and, where appropriate, major metabolite concentrations in plasma.In studies needed to support a future New Drug Application (NDA) fromthe Federal Drug Administration (FDA) for a prodrug such as IPdR,critical information for the application will be the rate of drugabsorption of IPdR by measuring plasma levels, the rate of conversion ofIPdR to IUdR using plasma measurements, and the rate of elimination ofIPdR, IP, IUdR and IU by metabolic and excretory processes. As IPdR isdesigned to be given at least once daily over a period of several weeksduring RT or combined RT and chemotherapy (chemoRT), detailed analysesof changes in PK parameters with the dosing schedule and duration ofdosing will be necessary to attempt to maximize tumor radio- (or chemo-)sensitization and/or minimize normal tissue toxicities. Additionally, PKinformation regarding demographic characteristics (age, sex, race),external factors (e.g. meals or other drug-drug PK interactions), anduse in specific cancer patient populations (e.g. high-grade gliomas,head and neck cancers, rectal cancers) will be collected in the clinicaltesting of IPdR. Detailed PK studies in several animal species werecompleted for FDA IND approval.

Extraction from the plasma and subsequent analysis by high performanceliquid chromatography (HPLC) of IPdR and its major metabolite,5-iodo-2-pyrimidinone (IP), and of IUdR and its major metabolite,5-iodouracil (IU), have been described by Kinsella, et al. An in vivocomparison of oral 5-iodo-2′-deoxyuridine and5-iodo-2-pyrimidinone-2′-deoxyribose toxicity, pharmacokinetics, and DNAincorporation in athymic mouse tissues and the human colon cancerxenograft, HCT-116. Cancer research. 1994; 54(10):2695-700. Thesenucleoside analogs are extracted from the plasma by the addition of5-chloro-2′-deoxyuridine (internal standard) and acetonitrile. Thesamples are redissolved in deionized water for HPLC analysis using aSpectra-Physics P2000 pump and UV2000 detection (Spectra-PhysicsAnalytical, Fremont, Calif.) on a 3.9×300 mm μ Bondapak C₁₈ reversephase column (Waters Associates, Milford, Mass.). Peaks are detected at335 mm (IPdR and IP) and 290 mm (IUdR and IU) versus authenticstandards. Typical retention times for IPdR, IP, IUdR, and IU were 21.9,14.0, 14.4, and 8.3 minutes, respectively. Seventy percent recovery ofthe nucleoside analogs is achieved using this method for all in vivostudies of IPdR prior to 2008. Since that time, a more sensitive assayof these plasma nucleosides has been established using liquidchromatography with tandem mass spectrometry (LC-MS/MS) (AgilentTechnologies, Santa Clara, Calif.) with the lower limits of quantitationof 0.1 μmol/L for IUdR and IPdR, and 0.25 μmol/L for IU and IP, asdescribed by Kinsella et al., “Toxicology and pharmacokinetic study oforally administered 5-iodo-2-pyrimidinone-2′deoxyribose (IPdR)×28 daysin Fischer-344 rats: impact on the initial clinical phase I trial designof IPdR-mediated radiosensitization,” Cancer Chemother Pharmacol. 2008;61(2):323-34. This more sensitive assay will be used in the proposedhuman clinical trials described in this invention. A sensitive liquidchromatography coupled with tandem mass spectrometry detection(LC/MS-MS) method can be used to measure plasma concentrations of IPdR,IdUrd, and other metabolites. Plasma samples are processed by solventdeproteinization. Separation of IPdR and its metabolites can beconducted on an Agilent 1200LC system (Agilent Technologies) using a4.6×250 mm Synergi Hydro-RP C18 column. All solvents should behigh-pressure liquid chromatography (HPLC) grade. All other reagentswill also be obtained from the Sigma-Aldrich Company. Calibration curveswill be constructed by adding known amounts of IPdR, IP, IdUrd, and IUrato control human plasma to give samples containing concentrationsranging 0.1 to 50 mmol/L of each compound. The response factor is linearover the range of 0.1 to 50 mmol/L. Samples will be diluted 1:10 incontrol matrix and reanalyzed when calculated concentrations exceed 50mmol/L. Kumar et al., “First-in-human phase 0 trial of oral5-iodo-2-pyrimidinone-2′-deoxyribose in patients with advancedmalignancies,” Clinical Cancer Research 2013; 19(7):1852-57.

In the prior clinical Phase I and II trials of continuous intravenousinfusions of IUdR+RT for high-grade gliomas (Table 1), high-gradesarcomas (Table 2), in patients with liver metastases for colorectalcancer, and in patients with head and neck cancers, the % IUdR-DNAcellular incorporation was measured in tumor tissue as well as normaltissues, including normal liver and in circulating blood cells(granulocytes). In general, the % IUdR-DNA cellular incorporation intumors and normal tissues correlated with the duration of the continuousIUdR infusions. In a clinical study of prolonged (28 days) continuousintravenous infusions, the % IUdR-DNA cellular incorporation incirculating granulocytes predicted for IUdR-mediated myelosuppression.In the pre-clinical studies comparing continuous IUdR infusions+RT anddaily po IPdR+RT, the % IUdR-DNA cellular incorporation was measured inhuman tumor xenografts, as well as in two normal tissues (bone marrowand bowel epithelium). Using % IUdR-DNA cellular incorporation as asurrogate of tumor radiosensitization in the xenografts and as asurrogate of normal tissue toxicity (e.g. weight loss,myelosuppression), it was found that IPdR was a more effectiveradiosensitizer and was associated with less normal tissue systemictoxicities compared to continuous infusion IUdR. This inventionvalidates this surrogate (i.e. % IUdR-DNA cellular incorporation) as ameasure of the TI of IPdR.

Two standard laboratory techniques (flow cytometry and high performanceliquid chromatography) have been established to measure the % IUdR-DNAcellular incorporation in tumor cells and normal cells, including cellsfrom the bone marrow, gastrointestinal epithelium, and circulatinggranulocytes. Flow cytometry is a laser-based, biophysical technologyemployed for marker detection (i.e. IUdR-DNA cellular incorporation) bysuspending cells in a stream of fluid and passing them by an electronicdetection apparatus. Fluorescence-activated-cell-sorting (FACS)(Becton-Dickinson, Mountain View, Calif.) is a specialized type of flowcytometry that allows for sorting a heterogeneous mixture of cells (e.g.cells with or without IUdR-DNA incorporation) based on the specificlight scattering and fluorescence characteristics of each cell. Tomeasure the proportion of cells “labeled” (i.e. with a defined %IUdR-DNA incorporation level) following an exposure to IPdR, the tumorand normal tissue/cells from the patient are obtained by needle biopsy(or phlebotomy for circulating granulocytes) and processed by mechanicalmincing and pepsin digestion for tumor specimens, or Ficoll-Hypaqueisolation followed by a Histopaque density gradient (Sigma Chemical Co.,St Louis, Mo.) for granulocytes, to obtain a single-cell suspension.After fixing the single-cell suspension with 70% ethanol in normalsaline, the DNA is partially denatured with 2N HCl and subsequentlyreacted with a mouse anti-IUdR monoclonal antibody (Becton-Dickinson)followed by incubation with a fluorescein isothiocyanate-labeled goatanti-mouse secondary antibody. Finally, the nuclei are incubated withpropidium iodide (PI) to label the DNA. Flow cytometric analysis isperformed using two color analysis with emissions of fluoresceinisothiocyanate detected with 530 nm short pass filters and PI detectedwith 630 nm long pass filters. In various embodiments, a minimum of10,000 events is analyzed per sample, and the percentage of labeledcells is calculated.

As used herein, the second technique to measure the % IUdR-DNA cellularincorporation as a surrogate for IPdR-mediated tumor radiosensitizationfor “difficult to cure” human cancers involves the use of highperformance liquid chromatography (HPLC). HPLC is a technique inanalytic chemistry that is used to separate the components in a mixture,to identify each component, and to quantify each component. It relies onpumps to pass a pressurized liquid solvent containing the sample mixture(e.g. cellular DNA exposed to IUdR) through a column filled with a solidabsorbent material. To measure the % IUdR-DNA cellular incorporationfrom tumor biopsies or circulating granulocytes taken from patientsreceiving po IPdR, single-cell suspensions are obtained similar to theprocessing for flow cytometry described above. Subsequently, singlecells are disrupted and the DNA is enzymatically digested into freenucleosides. DNA extraction and digestion are performed by incubationwith 10% trichloroacetic acid, followed by RNA digestion in 0.25M NaOHand enzymatic digestion with DNA-ase, alkaline phosphate andphosphodiesterase in MgCl and potassium phosphate buffer. A Waters 600Esolvent delivery system on a 3.9×300 mm μ Bondapak reverse-phase column(Waters Corp, Milford, Mass.) is used. The mobile phase consists of 100mm of sodium acetate buffer (pH 5.45) plus 7% (v/v) acetonitrile. Peakidentification and quantitation of IUdR and thymidine (TdR) areperformed against authentic nucleoside standards. The % IUdR-DNAreplacement (or % TdR replacement) is calculated according to:

$\frac{\left\lbrack {{mMoles}\mspace{14mu} {IUdR}} \right\rbrack \times 100}{\left\lbrack {{{mMoles}\mspace{14mu} {IUdR}} + {{mMoles}\mspace{14mu} {TdR}}} \right\rbrack}$

The therapeutic index (TI) of a drug is a quantitative relationshipbetween the safety (toxicology) of a drug and its efficacy(pharmacology). A drug's TI is a ratio between two doses, the dose ofthe drug that causes adverse effects at an incidence/severity notcompatible with the targeted indication divided by the dose that leadsto the desired pharmacological effect. The actual TI (i.e. value), then,is wholly dependent upon the choice of doses used to calculate theratio. Those choices are not standardized, as they necessarily varydepending upon the clinical situation. Historically, TI was determinedin animals using the lethal dose of a drug for 50% of the population(LD50) divided by the minimum effective dose for 50% of the population(ED50), resulting in the TI=LD50/ED50; and in humans, safety was definedas the toxic dose in 50% of patients (TD50), and efficacy was defined asthe efficacious dose in 50% of patients (ED50), and the TI for a drugwas calculated as: TI=TD50/ED50. Even this definition still requireschoices as to what is considered “toxic” and what is considered“efficacious.” Modern pharmacology now qualifies the historicaldefinition by recognizing that toxicity must be viewed in the context ofthe targeted indication (i.e. greater toxicity is acceptable if theindication is treating a lethal disease rather than a minor skin rash),and efficacy also must be defined for the specific circumstance (i.e. onthe spectrum of any response to complete response). Even for a specificdisease, the TI will vary depending upon the condition of the patientsreceiving the drug (e.g. older or debilitated patients will suffer moretoxicities than healthy young patients). Because of the challengesinherent in making these choices for any given situation, TI is used asa concept rather than as an actual value. Drugs are broadly consideredto have high or low TI's, and are compared on these relative scales. Ahigh TI is obviously preferable, indicating that relatively littletoxicity is seen at doses that achieve the desired response. Oncologicdrugs, however, often have very low TI's (even <1, where toxicity isevident at subpharmacologic doses), and their use is justified by theirindication, i.e. treating a lethal disease. Despite it's limitations, TIis a useful concept to consider when developing or evaluating a drug,because it has implications in terms of feasibility (i.e. can the drugbe tolerated at a dose that effects any desired response) andapplicability (i.e. is the drug so toxic that it's use is limited tolethal diseases or tolerated well enough to be appropriate for minorindications).

In preclinical (i.e. animal) studies of IPdR, no deaths were seen, evenat very high doses (one dose/day×14-28 days), so the LD50 was neverreached. Furthermore, these doses produced pharmacologic drug levels inthe targeted tumor tissues (as measured by the % IUdR-DNAincorporation). Therefore, the TI of IPdR in animals was very high(technically, ∞ (infinity)) because the LD50 was never reached). Theactive drug, IUdR, is more toxic, but again, the LD50 was never reachedin animal studies of IV IUdR. The mechanisms involved in the metabolismof IPdR have very favorable implications with respect to the TI of IPdR.IPdR must be converted to an the active drug IUdR, and this conversionoccurs preferentially in liver and tumor cells compared to many othernormal cells by virtue of the fact that many normal cells (specifically,bone marrow and gastrointestinal mucosal cells) do not possess theenzyme, aldehyde oxidase, to convert IPdR to the active drug. Thisprofile, with minimal toxicity at pharmacologic doses makes IPdR anattractive drug for a variety of clinical indications. For example, IPdRmay be added to regimens that are already being administered at theirmaximally tolerated dose (MTD) without the need to reduce doses ofeither IPdR or drugs within the regimen. Of utmost significance is thefact that the TI of IPdR predicts that it can be safely administered atpharmacologic doses in conjunction with radiation therapy (RT) deliveredat different total RT doses and fractionation schedules.

The halogenated thymidine (TdR) analogs, bromodeoxyuridine (BUdR) andiododeoxyuridine (IUdR), are a class of pyrimidine analogs that havebeen recognized as potential radiosensitizing agents since the early1960s. Their cellular uptake and metabolism are dependent on the TdRsalvage pathway where they are initially phosphorylated to themonophosphate derivative by the rate-limiting enzyme, thymidine kinase(TK). After sequential phosphorylation to triphosphates, they are thenused in DNA replication, in competition with deoxythymidine triphosphate(dTTP), by DNA polymerase. Indeed, DNA incorporation is a prerequisitefor radiosensitization of human tumors by the halogenated TdR analogs,and the extent of radiosensitization correlates directly with thepercentage TdR replacement in DNA (i.e. % IUdR-DNA incorporation). Themolecular mechanisms of radiosensitization are most likely related tothe increased susceptibility of TdR analog-substituted DNA to thegeneration of highly reactive uracil free radicals by ionizing radiation(IR), which may also damage unsubstituted complementary-strand DNA.Repair of IR damage may also be reduced by pre-IR exposure to theseanalogs.

Over the last 30 years, there has been renewed interest in thesehalogenated TdR analogs as experimental radiosensitizers in selectedcancer patient groups. These analogs are rapidly metabolized in bothrodents and humans, principally with cleavage of deoxyribose andsubsequent dehalogenation by hepatic and extrahepatic metabolism, whengiven as a bolus infusion with a plasma half-life of <5 min.Consequently, prolonged continuous or repeated intermittent druginfusions over several weeks before and during irradiation arenecessary, based on in vivo human tumor kinetics, to maximize theproportion of tumor cells that incorporate these analogs during the Sphase of the cell cycle.

In the early 1980's, clinical testing of halogenated pyrimidines asradiosensitizers focused on patients presenting with high grade gliomasand sarcomas (Tables 1 and 2), although definitive phase III testing hasnot been performed. The magnitude of radiosensitization correlatesdirectly with the % IUdR-DNA cellular replacement and determination of %IUdR-DNA cellular incorporation in tumor cells can serve as a predictiveradiosensitization assay. Additionally, in small series of patients withhead and neck cancers or liver metastases from colorectal cancer, the %IUdR-DNA cellular incorporation in tumors ranged from 5-8%, but was lessthan 1% in adjacent normal liver tissue, further supporting atherapeutic window for IUdR-mediated radiosensitization. Although IUdRhas clear potential as a clinically active radiosensitizer, itsdevelopment has been limited by the need for prolonged continuousinfusion (ci), intra-arterial or intravenous, before and during RT toradiosensitize tumors. Prolonged ci of IUdR resulted in myelosuppressionand acute GI toxicities, limiting the tolerated doses and the potentialfor clinical radiosensitization. Consequently, NCI or pharmaceuticalcompanies did not pursue further clinical development of IUdR.

The pyrimidinone nucleosides, including5-iodo-2-pyrimidinone-2′-deoxyribose (IPdR, were initially developed asantiviral agents, based on the hypothesis that nucleosides without anamino group or oxygen at position 4 would be substrates for viral butnot mammalian TK. IPdR was found to have significant activity in herpessimplex virus-infected HeLa cells in vitro and in vivo following poadministration without toxicity to uninfected cells or mice. IPdR hasnever been tested as an antiviral in humans. Although initial studiessuggested that po IPdR did not require metabolism to IUdR for antiviralactivity, subsequent studies demonstrated an aldehyde oxidase, which ispresent in both rat and mouse liver, that efficiently converts IPdR toIUdR. Saif et al., “IPdR: a novel oral radiosensitizer,” Expert OpinInvestig Drugs. 2007; 16(9):1415-24. Other normal tissues in the rat andathymic mouse including intestine, bone marrow, lung and kidney show≧10-fold less activity of IPdR aldehyde oxidase. These findings led to ahypothesis that IPdR might increase the percentage IUdR-DNAincorporation and subsequent radiosensitization of activelyproliferating primary or metastatic tumors in liver, while minimizingdrug toxicity and/or radiosensitization to the adjacent quiescent normalliver parenchyma and possibly other rapidly proliferating normaltissues, including bone marrow and intestine. Dr. Cheng was issued aU.S. patent based on this hypothesis, entitled, “Determination ofprodrugs metabolizable by the liver and the therapeutic use thereof”(U.S. Pat. No. 5,728,684) on Mar. 17, 1998.

In an initial publication regarding IPdR from Dr. Kinsella's laboratory,this hypothesis was tested using an athymic mouse model, in which ahuman colon cancer cell line (HCT-116), was established as a xenograftin subcutaneous (sc) flank tissue and as liver metastases using anintrasplenic implantation technique. IPdR was tolerated as a daily pobolus (via gastric gavage) for 6 days at a dose up to 1 g/kg/d, whereaspo IUdR resulted in significant systemic toxicity (>10% body weightloss) at 250 mg/kg/d for 4 days. Pharmacokinetic analyses of po IPdRdemonstrated efficient metabolism of IPdR to IUdR, with peak plasmalevels of IUdR up to 45 μM within 15 min following bolus administrationof 250 mg/kg IPdR. A 2-3 fold increase in the % IUdR-DNA cellularincorporation in tumor was found with IPdR at 1 g/kg/d for 6 dayscompared to the MTD of po IUdR (250 mg/kg/d for 4 days), but nodifferential effect of po IPdR on % IUdR-DNA incorporation was notedbetween liver tumor metastases and sc tumor. However, comparing the %IUdR-DNA cellular incorporation in two proliferating normal tissues(bone marrow and intestine), significantly less (≧2-fold) % IUdR-DNAincorporation was found with po IPdR than was found with po IUdR,whereas % IUdR-DNA incorporation was <1% in normal liver with eithercompound. Thus, initial in vivo results comparing % IUdR-DNAincorporation in tumors to proliferating normal tissues suggested thatpo IPdR has a potentially greater therapeutic index for IUdR-mediatedradiosensitization of human tumors than does po IUdR.

In more recent pre-clinical studies using po IPdR, an improvedtherapeutic gain was documented for in vivo human tumor xenograftradiosensitization in athymic mice using daily po dosing of IPdR×6-14days during RT compared to continuous intravenous (ci) infusion IUdR forsimilar time periods using maximum tolerated dose schedules of IUdR. Theuse of ci IUdR mimicked the route of administration used in the clinicaltrials of IUdR that demonstrated IUdR's potential for clinicalradiosensitization (Tables 1 and 2). Using two different human coloncancer cell lines (HT-29 and HCT 116) and one human glioblastoma cellline (U251) as subcutaneous (sc) xenografts in athymic mice, we againfound ≧2-fold increases in % IUdR-DNA tumor cell incorporation and≧2-fold decreases in % IUdR-DNA cellular incorporation in proliferatingdose-limiting normal tissues (bone marrow and intestine) following poIPdR compared to ci IUdR. Additionally, using a tumor regrowth assay toassess response to RT, we found a 1.3-6.0 fold sensitizer enhancementratio (SER) (time to regrow to 300% initial tumor volume) with IPdR(qd×6-14 d) plus fractionated RT (2 Gy/d×4 d) in two human colorectal(HT-29 and HCT 116) and two glioblastoma xenografts (U251 and U87)compared to fractionated RT alone. Less (≦1.1) enhancement of the RTresponse was found using ci IUdR×6-14 d plus fractionated RT in thesehuman tumor xenografts. Based on prior studies, the calculated SER forpo IPdR of 1.3-1.5 would be predicted to result in clinically relevantradiosensitization of resistant human cancers.

To better explain this improved therapeutic index for human tumorradiosensitization using IPdR, we further investigated thepharmacokinetics of po IPdR using once daily dosings for 14 or 28 daysin rodents. As demonstrated in our first po IPdR study, there was rapidand efficient conversion of IPdR to IUdR by a hepatic aldehyde oxidasein athymic mice. Plasma levels of IPdR and IUdR appeared to peak within10-20 min of a po IPdR bolus. Plasma levels of IUdR decreased morerapidly than did IPdR over the first 90 min. Plasma levels of the othermetabolites, IP and IU, reached a peak within 45-90 min following a pobolus of IPdR.

We also found that plasma levels of IPdR remain elevated at 45 and 90min following po administration at the highest dose (1500 mg/kg).Although the persistent plasma levels of IPdR following 1500 mg/kgsuggested a partial enzyme saturation of hepatic aldehyde oxidase, theprolonged IPdR plasma levels may also result from the fact that thishighest dose required a slower bolus administration by gastric gavageover 15-20 min to reduce the risk of aspiration.

In another plasma PK study, we used once daily po IPdR dosing of 0.2 or1.0 gm/kg/day (equivalent to 1200 or 6000 mg/m2)×28 days in Fischer-344rats. This study was requested by the FDA as part of the pre-clinicaltesting of po IPdR prior to IND submission. Forty rats (10/sex/dosagegroup) were randomly assigned to the 2 dosage groups with multiple bloodsamplings on days 1 and 28, and single blood sampling prior to po IPdRon days 8 and 15. The IPdR and IUdR plasma concentration time profileswere evaluated using compartmental analysis modules andnon-compartmental analysis, respectively. Absorption of po IPdR was notdependent on sex, dose, or single (Day 1) versus multiple daily dosings(Day 28). After single doses of 1.0 gm/kg/day, maximal concentrations ofIPdR were achieved at 90 min after dosing. With increasing daily podosing, Cmax values for IPdR were less than dose proportional and AUCvalues for IPdR after 28 days were less than those observed after Day 1.The mean elimination half-life for IPdR at the highest dose was ˜5hours. IUdR concentrations in these rats showed no sex differences,peaking by 15 min after dosing and AUC values were ˜proportional withincreasing dose. The mean half-life for IUdR was 6 and 8 hours for malesand females, respectively. Thus, the PK profile for po IPdR appearsquite similar in mice and rats.

Using cytosolic extracts from normal human liver specimens, we foundthat normal human liver has significant IPdR oxidase activity thatresults in a rapid (15 minute) conversion of IPdR to IUdR. The humanliver IPdR-aldehyde oxidase was cytosolic, protein dependent, cofactorindependent, and inhibited by low concentrations of menadione andisovanillin but not allopurinol. Menadione and isovanillin are selectiveinhibitors for aldehyde oxidase, and allopurinol is a selectiveinhibitor for xanthine oxidase. These results indicate that aldehydeoxidase but not xanthine oxidase is involved in the conversion of IPdRto IUdR in human liver. Moreover, the cytosolic localization and thelack of cofactors required for IPdR oxidase activity is consistent withaldehyde oxidase, which is a flavin adenine dinucleotide-containingenzyme that is oxidized by molecular oxygen but not by 3-NAD⁺ or otherelectron-transferring enzymes.

In contrast to human liver, we found that human small intestine hadsignificantly lower (≧1 log) IPdR oxidase activity that was notinhibited by isovanillin or allopurinol, but was stimulated bymenadione. These results indicate that human intestine cytosol has someIPdR oxidase activity; however, this activity cannot be assigned toaldehyde oxidase or xanthine oxidase, and the importance of thisactivity and the enzyme system responsible for this activity remain tobe determined. In addition, the activity of aldehyde oxidase in smallintestine is considerably lower (>10-fold) than that found in liver whenassayed with 6-methlypurine and would be expected to have lesscontribution to IPdR oxidase activity. We also confirmed that IPdRoxidase activity is not detectable in HT29 human tumor xenografts or inliver metastases obtained from two patients with colorectal cancer.

Orally administered IPdR appears to have limited systemic toxicities inrodents. Our first dose escalation study involved daily po bolus IPdR×6days over the range of 500-2000 mg/kg/d. Using a change in thepercentage body weight during treatment as an index of systemictoxicity, athymic nude mice tolerated the highest doses of IPdR (2000mg/kg/d) with ≦10% body weight change. In comparison, athymic micetolerated ci IUdR at 50 mg/kg/d for 6 days with 10-15% weight loss butexperienced ≧20% weight loss by day 6 using 100 mg/kg/d.

In a second study, we evaluated the systemic toxicity of IPdR given as adaily po bolus for 14 days at either 750 or 1500 mg/kg/d in athymicmice. Using a change in the percentage body weight during drug treatmentand for up to 28 days after treatment as an index of systemic toxicity,we found essentially no change in the percentage body weight gain forthe 6 weeks of observation during and after drug treatment for the twoIPdR groups compared to the control group receiving similar volumes ofsterile water for 14 days by repeated daily oral gavage. Additionally,no other adverse effects were recorded regarding animal activity andbehavior in the three groups of athymic mice (control, 750 or 1500mg/kg/d) by daily observation. As such, we did not reach a MTD of IPdRusing this 14-day daily po bolus schedule in athymic mice. No furtherdose escalations were used because of the increased risk of pulmonaryaspiration with larger drug volumes (22000 mg/kg/d).

We used the Marshall Farms ferret (North Rose, N.Y.) as a non-rodentspecies for IPdR systemic toxicity and toxicology testing as required bythe FDA. For this IPdR systemic toxicity and toxicology study, thetwenty-four male or female ferrets were randomly assigned to four IPdRdosage groups receiving 0, 15, 150, and 1500 mg/kg/d by oral gavage×14days prior to sacrifice on study day 15. No RT was administered to theferrets. The dosage range and schedule of dosing were based on our priorpre-clinical studies of IPdR in athymic mice. All ferrets survived the14-day treatment. Ferrets receiving 1500 mg/kg/d showed observablesystemic toxicities with diarrhea, weight loss, and decreased motoractivity beginning at days 5-8 of the 14-day schedule. Overall, bothmale and female ferrets receiving IPdR at 1500 mg/kg/d experiencedsignificant weight loss (9% and 19%, respectively) compared to controlfollowing the 14-day treatment. No weight loss or other systemictoxicities were observed in other IPdR dosage groups. Grossly, noanatomic lesions were noted at complete necropsy, although liver weightswere increased in both male and female ferrets in the two higher IPdRdosage groups. Histologically, IPdR-treated animals showeddose-dependent microscopic changes in liver consisting of minimal tomoderate cytoplasmic vacuolation of hepatocytes which either occurred inthe peri-portal area (1500 mg/kg/d dosage group) or diffusely throughoutthe liver (lower dosage groups). Female ferrets in the highest IPdRdosage group also showed decreased kidney and uterus weights at autopsywithout any associated histological changes. No histological changeswere found in CNS tissues. No significant abnormalities in blood cellcounts, liver function tests, kidney function tests or urinalysis werenoted following the 14-day treatment.

A second toxicology study of po IPdR used a once daily×28-day dosingschedule in male and female Fischer-344 rats. Again, no RT wasadministered. Eighty male and female rats (10/sex/dosage group) wererandomly assigned to dosage groups to receive either 0, 0.2, 1.0 or 2.0gm/kg/day×28 days and one-half were observed to day 57 (recovery group).Animals were monitored at least once daily and weighed every 3-4 daysduring the IPdR dosing period and then weekly to day 57. Blood testing,including a CBC with differential and a comprehensive chemistry panel,was performed on Days 0, 8, 15, 29, 43 and 57. Five male and five femalerats from each dosage group underwent full necropsy on either Day 29 or57 of the study. Rodent housing and feeding guidelines were similar toour prior toxicology study in ferrets.

IPdR-related effects were clinically evident at 1.0 and 2.0 gm/kg/daygroups as indicated by observation of hunched posture, rough coat orthin appearance. The 2.0 gm/kg/day males experienced significantdecreases in mean body weights (8-11%) beginning on day 5 and continuingthrough the 28 day treatment period; but the weight loss was readilyreversible once treatment was completed. No drug-related deaths occurredand only one animal (male, 2.0 mg/kg/day group) had a gross pathologyfinding, a small thymus gland with microscopic evidence of thymiclymphoid depletion. Importantly, no severe histopathologic changes werefound at the interim (Day 29) or final (Day 57) necropsies in anytissue/organ.

A first-in-human Phase 0 non-therapeutic trial of oral IPdR in patientswith advanced malignancies was published in 2013 from the Center forCancer Research, NCI. The objective was to determine whether the oralroute of administration of IPdR was a feasible alternative to continuousintravenous infusion of IUdR, as suggested from our pre-clinical studiescited above. A single po dose of IPdR was administered, and patientswere followed for 14 days for safety assessments. Dose escalations wereplanned (150, 300, 1200, and 2400 mg) with one patient per dose leveland 6 patients at the highest dose level. The starting dose of 150 mgwas based on 10% of the tolerable dose from a repeat-dose study in themost sensitive animal species, the ferret, as described above. Bloodsampling was conducted over a 24-hour period for pharmacokineticanalysis.

A total of 10 patients participated in the study and all patientstolerated the IPdR well with no drug-related adverse events, accordingto NCI Common Toxicity Criteria version 4.0. Plasma concentrations ofthe active metabolite, IUdR, generally increased as oral doses of IPdRescalated from 150 mg to 2400 mg. At the highest IPdR dose of 2400 mg,all 6 patients achieved peak IUdR plasma levels of 4.0+/−1.02 umol/Lafter 1.67+/−1.21 hours and IUdR plasma levels remained above 1 umol/Lfor 3-4 hours with a half-life of 1.5 hours.

This trial showed the ability of a small Phase 0 study to providecritical information for decision making regarding future development ofa novel radiosensitizing drug like IPdR. Adequate plasma levels of IUdRwere obtained to justify proceeding with further clinical development oforal IPdR in combination with radiation, as described by this invention.

Based on these extensive pre-clinical studies (summarized in Table 3)that led to the FDA IND, four conclusions were reached by the inventorthat served as guidelines for the design of the ongoing Phase I andPharmacokinetic clinical trials of IPdR+RT. First, orally administeredIPdR has been demonstrated to be an effective in vivo radiosensitizerusing four different human tumor xenografts (two colorectal cancer andtwo glioblastoma cell lines) in athymic mice. Compared to either po orci IUdR, a ≧2-fold increase in % IUdR-DNA tumor cell incorporation and a≧2-fold decrease in % IUdR-DNA incorporation in proliferatingdose-limiting normal tissues (bone marrow and intestine) is foundfollowing po IPdR×6-14 days. Greater tumor radiosensitization, using aregrowth delay assay of these human tumor xenografts in athymic mice,was also found with po IPdR (sensitizer enhancement ratios of 1.3-6.0)versus continuous infusion IUdR (sensitizer enhancement ratio of ≦1.1).Thus, po IPdR has a greater therapeutic index (TI) for IUdR-mediatedradiosensitization in human tumor xenografts in athymic mice compared tothe parent compound, IUdR. We did not establish a MTD in athymic micefor IPdR over the dose range used.

Second, in contrast to our studies in athymic mice in which nosignificant systemic toxicities were found with daily po IPdR doses of1500 mg/kg/d×6-14 days, we found significant weight loss (10-20% bodyweight) and gastrointestinal side effects in ferrets receiving 1500mg/kg/d×14 days and significant weight loss (˜10%) in male ratsreceiving 2 gm/kg/day×28 days. Mild to moderate microscopichistopathologic changes were noted in hepatocytes in an IPdRdose-dependent manner in ferrets and rats. No other gross or microscopicchanges were noted at complete necropsy. Additionally, no changes inblood counts, liver function tests, renal function tests or urinalysiswere found. While no toxic deaths were found in the ferret or ratstudies, we assigned the MTD for po IPdR at ≦1500 mg/kg/d×14 days and<2000 mg/kg/d×28 days.

Third, based on our pharmacokinetic data in Rhesus monkeys, rats, and inathymic mice, we conclude that oral or iv IPdR is rapidly cleared fromplasma in a bi-exponential fashion. However, it is also evident thatIPdR oxidase activity is partially saturable following high (>1000mg/kg) single doses in mice and following repeated daily doses over twoweeks in ferrets and over four weeks in rats. Collectively, these rodentand mammalian data of IPdR indicate the need for a careful, humanpharmacokinetic study of po IPdR as part of the initial Phase I clinicaltrials. We have already determined that normal human liver hassignificant IPdR-aldehyde oxidase activity. Human liver IPdR-aldehydeoxidase is cytosolic, protein dependent, and co-factor independent. Itis inhibited by low concentrations of menadione and isovanillin, but notallopurinol. Menadione and isovanillin are selective inhibitors foraldehyde oxidase while allopurinol is a selective inhibitor for xanthineoxidase. Thus, our pre-clinical results indicate that a human hepaticaldehyde oxidase, but not a hepatic xanthine oxidase, is involved in theconversion of IPdR to IUdR. It has been previously reported thatkinetically distinct forms of aldehyde oxidase exist in male and femalerodent liver, and that these distinct forms occur as a result ofdifferences in redox state and not in cDNA sequencing. However, a genderdifference in enzyme activity, as defined by in vitro conversion of IPdRto IUdR, was not evident in our studies in ferrets or athymic mice. Ahigh degree of homology exists between mouse and human aldehyde oxidase.

Fourth, based on the systemic toxicity data, the FDA recommended astarting po IPdR dose of 85 mg/m² (150 mg) qd×28 days (≅0.1 MTD) inhumans for the initial Phase I trials. The 28-day, once daily schedulewas chosen to provide adequate drug exposure prior to (one week) andduring (three weeks) RT to affect radiosensitization. Based on theobserved gastrointestinal toxicity seen in the ferret study and thehigher % IUdR-DNA levels in normal intestine cells compared to normalbone marrow cells found in our previously published IPdR studies inathymic mice, we are carefully monitoring patients for gastrointestinaltoxicity using the NCI Common Toxicity Criteria, version 4.0. Again, wehave already determined that human small intestine has significantlylower IPdR oxidase activity (≧10-fold reduction) compared to normalhuman liver. If IPdR can be safely administered in humans with favorablepharmacokinetics and increased % IUdR-DNA cellular incorporation intumors compared to dose-limiting proliferating normal tissues, it shouldgreatly reduce the cost and complexity of administration of ahalogenated thymidine analog for human tumor radiosensitization comparedto prolonged continuous intravenous or intra-arterial infusions asrequired for the parent compound, IUdR.

Many of the commonly used chemotherapeutic drugs, as well as RT, targetDNA for cytotoxicity. Indeed, the subsequent DNA damage response tothese cancer treatments in both malignant and normal tissues determinesthe therapeutic index (TI). The DNA damage response is a complex processinvolving multiple DNA repair, cell survival, and cell death pathwayswith both damage specificity and coordination of the DNA damage responseto different types of DNA damage. These DNA damages includedouble-strand breaks (DSB), single strand breaks (SSB), base damages,bulky adducts, intra/interstrand cross links, and breakdown ofreplication fork lesions.

The cytotoxic response to combining IPdR+RT is mediated through the DNAdamage response in both malignant and normal tissues. It is also nowunderstood that human cancers typically arise after a long process ofrandom gene mutations, particularly arising during repeated celldivisions of normal self-renewing (“stem”) cells that maintain normaltissue homeostasis. Some of these mutations that lead to cancer involvegenetic changes in key DNA repair pathways, including both DNA mismatchrepair (MMR) and base excision repair (BER). Additionally, epigeneticchanges via DNA methylation and acetylation at DNA repair genes can leadto cancer. As such, cancer treatments that target a specific DNA repairdefect can be selectively toxic (i.e. lethal) to cancer cells withdifferent DNA repair capacities while sparing normal (DNA repairproficient) cells. Based on pre-clinical cellular and molecular studiesby Dr. Kinsella's laboratory, it is known that specific IUdR-DNAmismatches (particularly G:IU mismatches) are recognized and repairedefficiently by both MMR and BER in normal cells. However, MMR-deficient(MMR⁻) and/or BER-deficient (BER⁻) cancers do not recognize nor repairthe G:IU DNA adducts caused by IUdR or IPdR treatment, resulting inincreased IUdR-DNA incorporation and enhanced IR cytotoxicity.Consequently, targeted treatment using IR+IPdR of MMR⁻ and/or BER⁻ humancancers will be exploited by this invention.

MMR is a highly conserved, but complex, DNA repair system that helpsmaintain genomic stability in human cells on several levels, includingcorrecting base-base mismatches and insertion-deletion loops (IDLs)erroneously generated during DNA replication. MMR also mediates cellcycle and cell death in response to certain types of endogenous DNAdamage and exogenous DNA damage from occupational and therapeuticchemical and ionizing radiation exposures. As such, MMR plays anessential role in the overall DNA damage response in humans by removingseverely damaged cells and reducing the risk of mutagenesis andcarcinogenesis. However, in the absence of MMR (i.e. MMR deficiency;MMR⁻), resulting from genetic and/or epigenetic alterations in the humanMMR genes, the persistent DNA base-base mismatches and IDLs remainingafter DNA replication result in a mutator phenotype with a 10²-10³elevation of spontaneous mutations highlighted by microsatelliteinstability (MSI) and a significant risk of cancer.

MMR deficiency is principally associated with the autosomal dominantLynch Syndrome, a consequence of mutations in MMR genes. MMR deficiencyis also associated with an increasing number of sporadic (non-genetic)common solid cancers, typically related to promoter methylation of thehMLH and hMSH2 genes. These sporadic MMR deficient human cancers includeseveral types of gastrointestinal cancers (colorectal, pancreatic,gastric, esophageal), gynecologic cancers (endometrial, ovarian),genitourinary cancers (bladder, ureter), as well as non-small cell lungcancers (NSCLC) and primary adult brain tumors, where MMR deficiency(detected by standard pathological immunohistochemistry (IHC) testing ofMMR protein levels in these tumors is found in up to 5-15% or more ofthese common cancers.

In pre-clinical (laboratory based in vitro/in vivo) studies ofMMR-deficient human cancer cells, resistance (“damage tolerance”) isfound to multiple different classes of clinically active chemotherapydrugs, including temozolomide, topotecan, cisplatinum, carboplatinum,5-fluorouracil (5-FU), and 6-thioguanine (6-TG), as well as to ionizingradiation. The clinical implications for the treatment of MMR-deficientsporadic human cancers are of both prognostic and predictivesignificance. For example, promoter hypermethylation of hMLH1 or hMSH2with subsequent loss of protein expression of these key MMR regulationproteins by IHC testing is found in nearly 50% of NSCLCs occurring innon-smokers, and is associated with a poor prognosis, even in earlystage disease. Additionally, recent analyses of multiple clinical trialsof the use of 5-FU (±concomitant cisplatinum or oxaloplatinum) asadjuvant treatment in MMR-deficient colon and esophageal cancers foundsignificantly less benefit in disease-free and overall survival incomparison to a significant benefit in MMR-proficient colon andesophageal cancers, respectively. MMR-deficient endometrial and rectalcancers also show reduced local control and lower pathological responserates following RT alone or with combined 5-FU and RT. Finally,MMR-deficient malignant gliomas (high grade adult brain tumors) werenoted to have a markedly reduced response rate and survival timecompared to MMR-proficient gliomas when treated with concomitant RT andtemozolomide.

MMR deficiency occurring during or following cancer treatment, relatedto MMR gene mutations or methylation/acetylation of the gene promoter,may also be associated with a poor prognosis. Somatic point mutations inMSH6 are found in up to 30% of recurrent/progressive glioblastomas,which were not present in pre-treatment specimens. Indeed, inactivationof MSH6 was correlated with prior or ongoing temozolomide exposure andassociated with enhanced tumor regrowth and shorter survival. Decreasedprotein expression of MLH1 following doxorubicin-based chemotherapy inbreast cancer patients was also reported to correlate significantly witha reduced disease-free survival (p=0.0025). Finally, promotermethylation of MLH in plasma DNA after cisplatin-based chemotherapy forovarian cancer predicted a poor survival.

Thus, these clinical data underscore the observed resistance (“damagetolerance”) to different classes of chemotherapy drugs alone, radiationtherapy alone, and chemotherapy-radiation therapy combined treatmentsfound in the pre-clinical studies in MMR-deficient vs MMR-proficienthuman cancer cells as mentioned above. This invention, IPdR+RT, isdesigned to overcome the “damage tolerance” of MMR-deficient cancers andwill be used clinically in different types of MMR-deficient cancers.

BER is the major DNA repair pathway involved in the removal of nonbulkybase damages induced by endogenous and exogenous adducts. A major sourceof endogenous base damage involves oxidative base modifications fromreactive oxygen and nitrogen species during normal cellular respirationor during oxidative stress from ischemia or chronic inflammation. BER isalso the major repair pathway for nonbulky damaged bases, abasic sites,and DNA SSBs after treatment with ionizing radiation, monofunctionalalkylating drugs (e.g. temozolomide), and certain antimetabolitesincluding the thiopurines (e.g. 6-thioguanine (6-TG) and6-mercaptopurine (6-MP)), the fluoropyrimidines (e.g. 5-FU), and thehalogenated thymidine analogues, IUdR and its prodrug, IPdR. Thus, BERand MMR pathways are activated by similar types of DNA damage-targetedcancer treatments and are involved in damage (sub) processing of bothIUdR and IR as pertains to this invention, as well as otherchemotherapeutic drugs (e.g. 6-thioguanine, 6-TG).

BER is a complex multistep pathway initiated by damage-specific DNAglycosylases, which create abasic or apurinic/apyrimidinic (AP) sites bycleaving the N-glycosidic bond and holding the base onto thesugar-phosphate backbone. Next, AP endonuclease 1 (APE 1) recognizes theAP sites and cleaves the DNA phosphodiester backbone leaving a3′-hydroxyl group and a 5′-deoxyribose phosphate group flanking thenucleotide gap. Subsequent repair proceeds by two subpathways, bothinitiated by DNA polymerase 13, for 1 nucleotide repair (short-patchBER) or for 2 to 15 nucleotides repair (long-patch BER). Although thesetwo subpathways use different subsets of enzymes, there is cooperationand compensation between the short-patch and long-patch pathways. It isgenerally held that short-patch BER accounts for most BER activity afterchemotherapeutic treatment and/or RT.

BER processing typically leads to chemotherapy drug and IR resistance.Consequently, the recognition and detection of BER-deficient cancersand/or the development of chemical inhibitors of specific BER enzymescan reverse alkylating and anti-metabolite drug resistance, as well asIR resistance. Based in part on the pre-clinical results from Dr.Kinsella's lab, methoxyamine (MX), a chemical inhibitor of BER, iscurrently undergoing clinical testing as TRC-102 in combination withchemotherapy drugs including temozolomide. MX is a small organic aminederivative of alkoxyamine that blocks the short patch BER pathway bycovalently binding the AP site formed by a specific glycosylase,rendering it refractory to the catalytic activity of AP endonuclease 1.A MX-bound AP site is repaired much more slowly (>300×) than a normal APsite, and enhances cell death. The Kinsella lab has also demonstratedthat the combination of IUdR+MX enhances % IUdR-DNA incorporation andradiosensitization in several experimental human cancer cell models.

Targeting the clinical use of IPdR as a radiosensitizing drug forMMR-deficient (damage tolerant) human cancers as part of this inventionwill also be extended to targeting BER deficiency in thesedamage-tolerant cancers with the use of methoxyamine (MX) or otherchemical APE1 inhibitors currently undergoing clinical development. Thehighlights of this targeted approach are as follows. First, from amolecular biochemical perspective, specific IUdR-DNA mismatches (i.e.,G:IU but not A:IU) are recognized and efficiently repaired by MMR.Consequently, MMR-deficient tumors retain significantly higher IUdR-DNAlevels compared with proliferating MMR-proficient normal tissues.Because the level of IUdR-DNA incorporation directly correlates with theextent of tumor radiosensitization, MMR-deficient human cancers can beselectively targeted to increase ionizing radiation cytotoxicity.Additionally, use of a chemical inhibitor of BER, such as methoxyamine(MX), will increase the % IUdR-DNA tumor cell incorporation, as the G:IUmismatch can also be processed (removed) by BER. This chemicalinhibition of BER will further increase the % IUdR-DNA tumor cellincorporation and subsequent IUdR-mediated radiosensitization.

Finally, BER-deficient cancers will be directly targeted by IPdR+RT aspart of this invention.

Second, using a probabilistic model of the cell cycle, faster cellcycling in MMR-deficient versus MMR-proficient cells is noted by theinventor, and from this, a computational model can predict when tumorcells with higher IUdR-DNA levels should be irradiated as tumor cellsaccumulate in more ionizing radiation-sensitive cell cycle phases.

Third, the therapeutic index for this treatment strategy can be assessedby quantifying IUdR-DNA incorporation levels in biopsy specimens ofMMR-deficient or BER-deficient tumors versus dose-limitingMMR-proficient and BER-proficient normal tissues (e.g. circulatinggranulocytes) by routine immunohistochemistry and flow cytometry withanti-IUdR antibodies and/or by high performance liquid chromatography(HPLC) approaches. Details of these techniques are previously providedunder DEFINITIONS.

Indeed, a proof-of-principle human tumor xenograft study in athymic micewas performed by Dr. Kinsella's lab, and showed persistently higherIUdR-DNA incorporation in MMR-deficient vs MMR-proficient tumor and a40% prolongation of tumor response with no dose limiting systemictoxicities.

Extensive preclinical data and limited clinical (Phase I and Phase I/II)studies (summarized in Tables 1 and 2) have demonstrated theradiosensitizing effect of ci IUdR in clinically radioresistant cancerssuch as high-grade gliomas and soft tissue sarcomas. These studies havealso demonstrated that IUdR is incorporated into DNA; this incorporationcan be reliably measured; and that the % IUdR-DNA cellular incorporationis directly and linearly related to the plasma level of IUdR (plasmalevel of >1 μmol/L to achieve >3% IUdR-DNA cellular incorporation).Despite these positive findings, further development of IUdR as aclinical radiosensitizer has not been pursued because the dose of IUdRthat is required to achieve plasma levels adequate forradiosensitization (i.e. >1 μmol/L to achieve >3% IUdR-DNA cellularincorporation) cause unacceptable normal tissues toxicity, and are thusnot tolerable (i.e. IUdR has an unacceptable therapeutic index). Inaddition, the required mode of administration of IUdR, namely continuousintravenous administration throughout an entire course of radiation ofup to 6 weeks, makes IUdR technically untenable to deliver in areasonable clinical setting. So, the addition of IUdR to the combinedmodality (i.e. RT+chemotherapy) approaches that now serve as thestandard of care for the treatment of many cancers has not been studied.

In contrast, IPdR, a prodrug of the radiosensitizer IUdR, is a novelorally available nucleoside analog that is predictably and efficientlyabsorbed in the GI tract and metabolized in the liver by aldehydeoxidase to IUdR. In addition to the advantage of ease of administration,the metabolism of IPdR to IUdR in vivo results in two additional majoradvantages of po IPdR compared to ci IUdR. First, compared to ci IUdR,oral IPdR generates 2-3 fold increased % IUdR-DNA incorporation intotumor tissues, and second, oral IPdR results in a 2-3 fold decreased %IUdR-DNA incorporation into proliferating normal tissues (bone marrowand intestine) as a consequence of the properties and biodistribution ofaldehyde oxidase, the enzyme that converts IPdR to IUdR. Thus, asopposed to ci IUdR, po IPdR can be delivered at a dose that is bothtolerable and that produces plasma levels of IUdR adequate for effectiveradiosensitization.

Adding po IPdR to RT or combined RT+chemotherapy treatment regimens,then, can be expected to be feasible, tolerable, and moreover, improvethe overall efficacy of these treatments for several solid adultcancers. The clinical use of IPdR+RT to target sporadic MMR-deficientand/or BER-deficient cancers is already discussed above. Additionally,IPdR may be particularly valuable as a component of the followingcombined modality regimens: for high-grade brain tumors,(IPdR+RT)±temozolamide (TMZ); for rectal and gastric cancers,(IPdR+RT)+fluoropyrimidines (e.g. 5-fluorouracil (5-FU), capecitabine);and for head & neck and cervical cancers, (IPdR+RT)+platinum analogs(e.g. cisplatin (CDDP)); and for pancreatic, gynecologic and head & neckcancers, (IPdR+RT)+ribonucleotide reductase inhibitors.

A brief description of IPdR+RT+chemotherapy combinations for clinicaluse include the following.

First, Improvement in the TI of RT+/−TMZ in high-grade brain tumors.High-grade brain tumors, including anaplastic astrocytomas (AA) (Grade 3of 4) and glioblastoma multiforme (GBM) (Grade 4 of 4) are highlymalignant tumors, currently not curable. Approximately 13,000 patientsare diagnosed with these diseases yearly in the U.S. These tumors rarelymetastasize outside of the brain, and typically recur locally followinginitial surgery (maximum safe resection) followed by concomitant RT+TMZ,and later, adjuvant TMZ. The median survival for these tumors is ≈3.4years for AA and 14-18 months for GBM. The clinical data for the priorclinical trials of RT+ci IUdR (from the 1980-1990's, prior to thepresent use of RT+TMZ) are previously reviewed in Table 1, and aresimilar to the median survival for the current use of RT+TMZ.Pre-clinical data on RT+po IPdR using two different human glioblastomaxenografts suggest further improvement in the therapeutic index (TI).

As currently used, TMZ is the most effective chemotherapy drug forpatients with high risk low grade glioma and high-grade gliomas, and isroutinely used on a daily basis during a typical 6-week course ofpost-operative RT, based on clinical Phase 2 and 3 trials.

However, the biochemical and molecular interactions of TMZ+RT are onlyadditive, not synergistic as is the IPdR+RT interaction as described indetail herein.

Over the last decade, the molecular analysis of high-grade gliomas hasprincipally focused on defining the methylation status of the promoterregion of MGMT as a prognostic marker of survival and as a predictiveassay of TMZ efficacy on the tumor. MGMT promoter methylation is foundin approximately 40% of high-grade gliomas in adults and 80% in the muchless common pediatric high-grade gliomas. In clinical trials of RT+TMZin adult GBM patients, RT+TMZ was more effective (5-month survivalbenefit to 15 months, similar to the prior RT+ci IUdR data as presentedin Table 1) compared to RT alone in patients whose tumors showedmethylated MGMT. No survival advantage was found using RT+TMZ comparedto RT alone in unmethylated MGMT GBM patients, representing 60% of allGBM patients. However, despite MGMT methylation status-directed therapyfor adding TMZ to RT, virtually all patients with high-grade gliomaswill progress locally in brain, and succumb to the disease. As such,newer (more novel) RT treatment options are sorely needed for high-gradegliomas.

Dysregulation of other DNA repair pathways, including both MMR and BER,are reported to contribute to the aggressive biology and poor prognosisof high-grade gliomas. In a recent comprehensive analysis of themessenger ribonucleic acid (mRNA) expression levels in 157 DNA repairgenes in two large, publically available, gene expression data sets from699 GBM tumors, the expression levels of a key BER glycolylase (APE1)and a key MMR protein (PMS2) were reported to be independent prognosticbiomarkers of survival following treatment with RT alone or TMZ alone.Based on this novel DNA repair prognostic index for GBM and otherhigh-grade gliomas, future clinical studies of the use of RT+IPdR wouldstratify for specific molecular subsets of high-grade gliomas withintact MGMT (i.e. unmethylated promoter) and deficient MMR.Alternatively, combinations of RT+IPdR+TMZ would be recommended forhigh-grade gliomas with methylation of the MGMT promoter and low APE1expression, representing deficient BER. In both clinical scenarios, theenhanced IPdR-mediated radiosensitization would be predicted to improvethe duration of survival by ≧50%, as reflected in the early trials of ciIUdR+RT (Table 1).

Second, improvement in the TI of RT+fluoropyridimine (FP)-basedchemotherapy as pre-operative adjuvant treatment in locally advancedrectal cancers. Adjuvant therapy for solid tumors, including locallyadvanced rectal cancer (=10,000 cases/yr in the U.S.), is designed tocure patients more often than surgery alone. The first principles ofcurative adjuvant therapy are to improve both local tumor control andreduce the development of systemic (metastatic) disease. Through themid-1980's, locally advanced rectal cancer, defined by tumor extensionthrough the rectal wall (called cT₃ or T₄ disease) or involvement oflocoregional pelvic lymph nodes (called N₁ or N₂ disease) was treatedsurgically with a cure rate of only 35-40% and a high risk of localrecurrence (25-35%) and developing metastatic disease. Over the lastthree decades, pre-operative RT+concomitant FP-based chemotherapy(initial continuous intravenous 5-FU and now twice daily pocapecitabine) followed by total mesorectal excision (TME) surgery hasimproved the overall cure rate up to 65-70% in this patient group.

It is now recognized that the pathologic tumor response to locallyadvanced rectal cancer following RT+FP-based chemotherapy is ofprognostic significance for predicting local control, disease-freesurvival (DFS) and overall survival (OS) with ten years of follow-up. Inthe 10-15% of patients whose resected tumors following pre-operativeRT+FP treatment showed no residual viable tumor cells (i.e. a completepathologic response (pCR)), 10-year DFS approached 90%. In contrast, thevast majority of similarly treated locally advanced rectal cancerpatients whose resected tumors showed intermediate tumor regression (65%of patients) or poor tumor regression (25-30% of patients) achievedlower 10-year DFS, 70% and 60%, respectively. Thus, the pCR ratefollowing pre-operative RT+FP treatment is a valid intermediate for thispatient group based on this study and others.

More recently, clinical trials for locally advanced rectal cancerpatients have attempted to improve the pCR rate by adding othercytotoxic chemotherapy drugs (e.g. oxaliplatin) or biologic agents (e.g.cetuximab, an epidermal growth factor inhibitor) during pre-operativeRT+FP without any improvement in pCR rate, but with increased normaltissue complications, representing a decrease in the therapeutic index(TI).

The biochemical and cellular interactions of RT+FP are felt to resultfrom inhibition of the enzyme, thymidylate synthetase (TS) by the FPmonophosphate metabolite, FdUMP, leading to decreased (or unbalanced)nucleotide pools necessary for DNA synthesis and decreased DNA repairfollowing RT damage. Intracellular IPdR monophosphate metabolite, IdUMPcan inhibit (by binding) TS leading to increased FP-mediatedradiosensitization as well as enhancing IPdR-mediated radiosensitizationsecondary to IUdR-DNA incorporation. Consequently, the use of po IPdRadded to the current standard-of-care pre-operative adjuvant therapy(i.e. RT+twice daily po capecitabine) is postulated to increase the pCRrate from 10-15% to 30-35% based on the IPdR+FP interactions and IPdR+IRinteractions found experimentally in human colorectal cancer cells. Suchan increase in the pCR rate should translate into a further 10-15%improvement in DFS for patients with locally advanced rectal cancer. Theconcept of testing this hypothesis in a Phase II clinical trial of poIPdR+RT+twice daily po capecitabine was favorably reviewed whenpresented in November 2014 to the NCI Rectal Cancer Radiotherapy WorkingGroup. Currently, a NCI-sponsored initial Phase I and PK clinical trialof po IPdR+RT for patients with metastatic gastrointestinal cancers,including rectal cancer (NCI Protocol #9882;http://www.cancer.gov/about-cancer/treatment/clinical-trials/search/results?protocolsearchid=14235539),with Dr. Kinsella as the Principal Investigator, is ongoing. Theclinical and PK data from this ongoing clinical trial will be used inthe design of a future Phase II clinical trial of IPdR+RT+twice daily pocapecitabine as pre-operative therapy in patients with locally advancedrectal cancer.

Third, improvement in the TI of RT+platinum (PA) compounds in head andneck cancers and cervix cancer. Platinum-based (PA) compounds form adistinct class of cytotoxic chemotherapy drugs characterized by theirunique metallic element. The initial PA drug, cis-platin(cis-diamminedichloroplatinum II) exerts its cytotoxicity by inhibitingDNA synthesis, as well as by inhibition of transcription elongation bycreating DNA intrastrand crosslinks. In vitro experiments in rodent andhuman cancer cells suggest greater than additive interactions of PA withIR, mediated by inhibition of DNA repair. A unique in vitro biochemicalinteraction of cisplatin and IUdR previously reported results inincreased DNA crosslinks and enhanced cytotoxicity in a human bladdercancer cell line. Specifically, the effect of increasing concentrationsof IUdR for 48 hours prior to a one-hour exposure to cisplatindemonstrates dose-modifying factors of up to 3.5 at 10% survival. Inaddition, a time course for formation of an IUdR-platinum (Pt) adduct iscompared to a deoxyguanine (dGua)-platinum (Pt) adduct that appearsslower, but is similar to that of a deoxycytidine-platinum adduct. Twospecific chemical structures of the IUdR-Pt adducts are proposed. Thus,these experimental data suggest that the proposed clinical use of RT+poIPdR with daily to weekly PA treatment would be expected to enhance theefficacy of PA adduct formation (and tumor cytotoxicity) and furtherenhance the well-established effect of RT in these common tumor sites ina greater than additive (i.e. synergistic) manner. Importantly, it ishypothesized that there would be no overlap of the known dose-limitingsystemic normal tissue toxicities of PAs, that include myelosuppression,renal toxicity, ototoxicity and peripheral neuropathy with theconcomitant use of po IPdR, based on the pre-clinical IPdR toxicologydata.

Clinically, the combination of RT+PA-based chemotherapy (typically,cis-platinum) has been demonstrated to be more effective than RT alonefor patients with locally advanced squamous cell carcinomas (SCC)arising from the head and neck (H&N; =15,000 cases/year in the U.S.), aswell as to the cervix (=12,000 cases/year in the U.S.), based onmultiple randomized clinical trials over the last 2+ decades. As such,RT+PA remains the standard-of-care for both disease sites, and futureclinical trials to further improve the cure rate for these two patientgroups will incorporate IPdR into these RT+PA regimens.

As a proof of principle for future use of po IPdR combined with RT+PAchemotherapy, the threshold of % IUdR-DNA tumor cell incorporation (23%)associated with in vivo radiosensitization has already been confirmed inprior clinical Phase I trials of ci IUdR (or BUdR)+RT for both patientswith locally advanced H&N SCC and cervical SCC.

Using ci IUdR at 1000 mg/M², a dose that produces plasma levels of =1mMol/L, IUdR-DNA tumor cell levels plateaued at 7.5-8% within 5 days,suggesting a 1.3% DNA-replacement/day in H&N SCC. A similar dosing of ciBUdR (1000 mg/M²/day×4 days, repeated weekly for 6 weeks) in patientswith locally advanced cervical cancer resulted in tumor cell IUdR-DNAincorporation of 5-8% on Day 5 following a 4-day ci treatment.Furthermore, this level of IUdR incorporation into tumor cells comparedfavorably to the 3-4% IUdR-DNA incorporation in adjacent normal rectalmucosa cells, demonstrating a 1.5-1.8-fold ratio of tumor to rectum. Byextrapolating to the pre-clinical and clinical Phase 0 trials of poIPdR, the higher transient plasma levels of IUdR (up to 4 μMol/L), aswell as the favorable tumor-normal tissue (bone marrow; bowelepithelium) % IUdR-DNA cellular incorporation ratios from the mousehuman tumor xenograft studies as previously described, should result inan improved TI for the use of po IPdR with RT+PA in locally advancedcervix cancer patients.

Fourth, improvement in the TI of RT+ribonucleotide reductase (RR)inhibiting chemotherapy drugs in pancreatic, gynecologic and head & neckcancers. RR inhibiting chemotherapy drugs, including hydroxyurea,gemcitabine, fludarabine, motexafin gadolinium and3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3AP, triapine) havebeen studied as potential radiosensitizing drugs for over three decadesin both the laboratory and clinic.

RR is the rate limiting enzyme in the synthesis and repair of DNA and isthe only enzyme responsible for the conversion of ribonucleotidediphosphates to deoxyribonucleotide diphosphates, the fundamentalbuilding blocks of DNA synthesis and repair. RR is a heterodimerictetramer comprised of two dimers. R1 (also called RR M1) is the largeregulatory subunit that is constitutively expressed through the cellcycle and can bind the nucleoside analogs gemcitabine and fludarabine.There are two smaller subunits called R2 (or RR M2) and a p53 induciblehomolog of the R2 subunit (known as p53R2) that can bind to the R1 dimerto form the active enzyme.

There are two major theories involving the role of RR in processingionizing radiation (IR) damage. The first, derived from experimentaldata, is that the R2 protein is upregulated following IR providing more(sufficient) deoxyribonucleotide triphosphates (dNTPs) for DNA repair ofIR-induced damage. The second theory involves upregulation of p53R2protein, again to result in increased dNTP pools for IR-induced damagerepair. It has also been found that the use of non-cytotoxic doses ofthe RR inhibitor hydroxyurea and clinically achievable levels of IUdR (2μM) resulted in a 2-fold increase in IdUTP pools and IUdR-DNAincorporation resulting in a greater that additive (approximately 1.75)radiation sensitizer enhancement killing of human bladder and cervicalcancer cells in vitro.

Clinically, these RR inhibitor chemotherapy drugs, particularlyhydroxyurea, gemcitabine and triapine have been administered during RTwith an improvement in the TI for locally advanced cervix, head & neckand pancreas cancers based on clinical Phase 2 and 3 trials.Additionally, the dose limiting toxicity with these RR inhibitors isprincipally myelosuppression, and in the preclinical IPdR toxicitystudies, significant myelosuppression was not encountered, so theaddition of IPdR to RR inhibitor therapies should be feasible andtolerable. Local tumor control with RR inhibitors+RT is improved over RTalone by only 10-30%. It is postulated that the addition of IPdR couldfurther improve local control as well as overall cure rates,particularly in cervix and head & neck cancers. Indeed, it is alreadydemonstrated in a small clinical trial that ci IUdR+RT results in a highlocal control rate of locally advanced head & neck cancers with %IUdR-DNA levels>5% following a 5-7 day ci IUdR infusion.

As part of this invention, an assay will be further developed clinicallyto predict the extent of IUdR-mediated radiosensitization by IPdR, aswell as tumor proliferation before and during RT, and the potentialnormal tissue toxicities from IPdR±RT in selected cancers as previouslydescribed. The percentage IUdR-DNA incorporation in a tumor cell (invitro) or tissue (in vivo) correlates directly with the extent ofradiosensitization. Pre-clinical in vivo testing of oral IPdR as aprodrug for IUdR-mediated radiosensitization, consistently found ≧2-foldincreases in the percentage IUdR-DNA tumor cell incorporation and≧2-fold decreases in percentage IUdR-DNA incorporation in proliferatingdose-limiting normal tissues (bone marrow and intestine) following oralIPdR compared to ci IUdR (using MTD schedules) in four different humantumor xenografts in athymic mice. In prior clinical Phase I trials of ciIUdR, the percentage IUdR-DNA incorporation in circulating granulocytesduring and following the IUdR infusion as a surrogate for aproliferating normal human tissue (bone marrow) were measured by highpressure liquid chromatography (HPLC) and/or by flow cytometry. Ingeneral, the percentage IUdR-DNA incorporation in circulatinggranulocytes increased linearly with a linear increase in steady-stateplasma levels of IUdR. Additionally, high (≧6%) % IUdR-DNA levels inperipheral granulocytes predicted for systemic bone marrow toxicity withci IUdR in humans. In the pre-clinical studies of oral IPdR qd×14 d inathymic mice and ferrets, the percentage IUdR-DNA incorporation did notincrease linearly with increasing daily po IPdR dosing, and was ≦2% incirculating granulocytes following treatment, and importantly, nochanges in blood counts seen in ferrets and rats, suggesting that bonemarrow toxicity may not be dose-limiting for orally administered IPdR inhumans. Indeed, in the toxicology studies of po IPdR daily×14-28 days inmice, rats, and ferrets, no myelosuppression was seen.

In human Phase I studies of ci IUdR, single tumor biopsies of high-gradegliomas, high-grade soft tissue sarcomas, H&N cancers, and livermetastases from colorectal cancers obtained at defined intervals duringci IUdR were used to correlate the % IUdR-DNA incorporation in tumor tosteady-state drug levels. Again, a linear relationship of the % IUdR-DNAincorporation in tumor tissue and the steady-state plasma IUdR level wasfound.

The following examples are provided for illustration.

EXAMPLES Example 1

A 32 year old female has a 12 cm soft tissue mass in the left pelvis.Biopsy reveals a high-grade soft tissue sarcoma.

The tumor is technically unresectable due to extension into thelumbosacral plexus, and RT is the mainstay of therapy and the onlychance for cure. IPdR will be administered to the patient in the form ofan oral drug (optimal dose of IPdR to be determined in ongoing clinicaltrials, predicted to be in the range of 0.1-50 gm/M2/day, preferably 2to 5 gm/M2/day). The patient will undergo RT (typically, 70-80 Gy totaldose delivered in 1.8-2.0 Gy fractions, 1 fraction/day, 5 days per week)1-4 weeks (preferably 1-2 weeks, typically 1 week) following theinitiation of IPdR. The patient will continue to take oral IPdRthroughout the course of RT, and will discontinue IPdR followingcompletion of RT. Given the radiation sensitizing effect of IPdR ontumor cells relative to normal tissue cells (i.e. increasing thetherapeutic index (TI) of RT), maximum tumor control/eradication withminimal normal tissue toxicity will be achieved. This patient isexpected to have an improved chance of survival at 2 years.

Example 2

A 58 year old male has a 6 cm mid-rectal mass with adjacent lymph nodeenlargement suggestive of N1 disease. Biopsy shows adenocarcinoma. Thediagnosis is Stage III rectal cancer (adenocarcinoma), andstandard-of-care therapy involves neoadjuvant (pre-surgery) chemotherapyand radiation (chemoRT), surgical resection, and potentially adjuvant(post-operative) chemotherapy. Neoadjuvant therapy is a criticalcomponent to this treatment regimen as it shrinks and sterilizes (killscancer cells) the tumor as much as possible, and thus, optimizes theresults of surgery. At the time of surgery, the resected tumor isexamined pathologically, and if there is no evidence of residual viabletumor cells, the person is deemed to have a pathologic complete response(pCR). If pCR has been achieved, the 5-year disease-free survival (DFS)(considered equivalent to cure) rate is 85% vs 65% for those who do notachieve pCR. Furthermore, persons with a pCR do not require further(i.e. adjuvant) chemotherapy. In addition to the providing the mechanismfor optimal (i.e. achieve pCR) surgery and its resultant improvement indisease-free and overall survival, neoadjuvant chemoRT also has thepotential to lessen the morbidity of the surgical procedure by shrinkingthe tumor and thus requiring a less extensive resection (this may evenobviate the need for a permanent colostomy in persons who would haverequired it if the surgery had been performed without neoadjuvantchemoRT).

The fluoropyrimidine class (FP) of chemotherapeutic agents including,5-fluorouracil, floxuridine, capecitabine, and DPD-inhibitingfluoropyrimdines, is known to be effective in colorectal cancertreatment. FPs reduce a cell's ability to repair DNA damage, includingthe damage caused by RT. This effect is mediated through inhibition ofthymidylate synthetase (TS), which then causes depletion/alteration ofthe nucleotide pools needed for DNA repair. The addition of IPdR furtherinhibits TS, with a greater than additive effect, as a result of thegeneration of intracellular IPdR monophosphate metabolite, IdUMP thatfurther inhibits (by binding) TS leading to increased FP-mediatedradiosensitization as well as enhancing IPdR-mediatedradiosensitization. For rectal cancer, capecitabine is used in theneoadjuvant setting, with concomitant RT, and other FPs are used inadjuvant regimens (typically, 5-FU as part of a FOLFOX (formic acid,5-FU, oxaliplatin) regimen). The use of neoadjuvant capecitabine+RTallows a pCR in approximately 10-15% of Stage III rectal patients. Theaddition of IPdR to capecitabine+RT is predicted to increase the pCRrate from based on the IPdR+FP interactions and IPdR+IR interactions.Such an increase in the pCR rate should translate into a furtherimprovement in 5-year DFS for patients with locally advanced rectalcancer.

IPdR will be given in conjunction with capecitabine+RT in the neoadjvantsetting for a person with Stage III rectal cancer, with the goal ofenhancing the degree of tumor reduction prior to surgery (improving thepCR (and thus, cure) rate and reducing the need for extensive (i.e.morbid) resection). Prior to the initiation of RT, IPdR will beadministered to the patient in the form of an oral drug (optimal dose ofIPdR to be determined in ongoing clinical trials, predicted to be in therange of 0.1-50 gm/M2/day, preferably 2 to 5 gm/M2/day). The patientwill begin capecitabine therapy (typically, 750-875 mg/M2/dose bid) 1-4weeks (preferably 1-2 weeks, typically 1 week) following the initiationof IPdR, and undergo RT (typically, 50.5 Gy total dose delivered in 1.8Gy fractions, 5 days per week, for 28 fractions). The patient willcontinue to take oral IPdR in addition to capecitabine therapythroughout the course of RT, and will discontinue IPdR and capecitabinefollowing completion of RT. The high therapeutic index of IPdR (minimaltoxicity encountered at clinically effective dose), and specifically thelack of gastrointestinal toxicity and hand-foot syndrome (the doselimiting toxicities of capecitabine are gastrointestinal toxicity andhand-foot syndrome)) allows addition of IPdR, with its radiationsensitizing effect, to the capecitabine+RT regimen without the additionof significant toxicity.

Three to six weeks following completion of IPdR+capecitabine+RT,surgical resection will be performed. It is predicted that thesynergistic radiation sensitizing effect of IPdR+capecitabine willenhance the pCR rate compared to capecitabine alone, so that morepatients receiving IPdR+capecitabine+RT will achieve pCR. In thisexample, the patient will undergo non-radical (i.e. rectum-sparing, notrequiring a permanent colostomy) resection, and pathology will revealpCR, thus the patient will require no adjuvant chemotherapy, and will beexpected to have a higher cure rate.

Example 3

A 62 year old male with a history of smoking has a 5 cm mass arisingfrom the left tonsil, bilateral regional lymph node involvement and noevidence of distant metastatic disease (large primary tumor (>4 cm; T3),regional lymph node involvement (N3), and no distant metastases (MO)disease; typical of most head & neck cancers). Biopsy reveals squamouscell histology. As a result of his smoking habits, the patientdemonstrates significant cardiac and pulmonary co-morbidities, renderinghim high risk, and thus a poor candidate, for anesthesia and anyextensive surgical procedure.

For cancers including those originating from the head & neck,gastrointestinal tract, genitourinary system, gynecologic system, lungand bone and soft tissue sarcomas, the platinum-based class (Pt) ofchemotherapeutic agents including, but not limited to, cis-platin,carboplatin and oxaliplatin, is known to be effective. Administration ofPt agents produces intracellular Pt-DNA adducts in these tumors that arethen unable to appropriately repair the additional DNA damage caused byRT. The addition of IPdR (IUdR) produces unique IU:Pt adducts in DNA,enhancing Pt cytotoxicity and greater than additive (i.e.synergistically) enhancement of the individual RT-sensitizing effects ofPt agents and IPdR (IUdR).

Given the patient's high risk for surgery, his treatment plan willinvolve a combination of chemotherapy and definitive RT (for thosepatients who are surgical candidates, a lower, neoadjuvant (i.e.pre-operative) dose of RT would be administered). Prior to theinitiation of RT, IPdR will be administered to the patient in the formof an oral drug (optimal dose of IPdR to be determined in ongoingclinical trials, predicted to be in the range of 0.1-50 gm/M2/day,preferably 2 to 5 gm/M2/day). After one week of IPdR therapy, thepatient will begin cis-platin (100 mg/M2 IV on days 1, 22 and 43 or40-50 mg/M2 q week×6-7 weeks) and RT (definitive dose, total dose: 66-74Gy administered in 2.0 Gy fractions, 1 fraction/day, 5 days/week×6-7weeks). Cis-platin and IPdR will be discontinued following completion ofRT. The high therapeutic index of IPdR (minimal toxicity encountered atclinically effective dose), and specifically the lack of renal toxicity,ototoxicity, peripheral neuropathy and myelosuppression (i.e. will notaugment the dose limiting toxicities of cis-platin (renal toxicity,ototoxicity, peripheral neuropathy and myelosuppression)), allowsaddition of IPdR, with its radiation sensitizing effect, to thecis-platin+RT regimen without the addition of significant toxicity.

Following completion of IPdR+cis-platin+RT, physical examination andimaging evaluation indicate no evidence of residual tumor, i.e. aclinical complete response (cCR). cCR is the goal ofchemotherapy+definitive RT, as it obviates the need for surgery toremove residual tumor. Here, the patient is spared the high risk ofsurgery to render him free of residual tumor. A patient who receivescis-platin+definitive RT and achieves a cCR has a cure rate ofapproximately 60%. The addition of IPdR to cis-platin+definitive RTresults in enhancement of cytotoxicity on the basis of both drug-drug(i.e. IPdR-Pt) and drug-drug-RT (IPdR-Pt-RT) interactions, and would bepredicted to further improve the patient's cure rate.

Example 4

A 37 year old male presents with headache and a seizure. MRI reveals a 4cm enhancing lesion in the left cerebral cortex with areas ofhemorrhage, peritumoral edema and lack of calcification. No additionalintraparemchymal lesions are noted. Biopsy reveals a glioblastomamultiforme (GBM), and molecular analysis of the tumor cells reveals MGMTpromoter methylation.

For specific cancers originating from the central nervous system andbone and soft tissue sarcomas, methylating agents (MA) including,temozolomide (TMZ) and the nitrosoureas are known to be effective.Specifically, in high-grade gliomas with MGMT promoter methylation (upto 40% of patients), TMZ produces an additive biochemical enhancement ofRT cytotoxicity. Clinical trials have demonstrated a 5-month survivalbenefit for the addition of TMZ to RT for patients with MGMT promotermethylated high-grade gliomas (from 10 months to 15 months), and so itis considered the standard-of-care, but virtually all patients willrecur within the same area of the brain. However, randomized Phase 3clinical trials show no survival benefit to TMZ+RT vs RT alone in GBMpatients whose tumors possess an unmethylated MGMT promoter (60% of allGBM patients). TMZ is administered with RT even for these unmethylatedMGMT high-grade gliomas because it is well tolerated and survival afterRT alone is so dismal that TMZ is given with the hope of deriving some,albeit statistically insignificant, benefit.

Clinical trials of continuous infusion (ci) IUdR+RT for patients withhigh-grade gliomas demonstrated a prolongation of survival of greaterthan 50% compared to RT alone (Table 1). This survival benefit of ciIUdR+RT compared to TMZ+RT is predicted based on the molecularinteractions of TMZ+RT, which are only additive, compared to themolecular interactions of IUdR+RT, which are greater than additive (i.e.synergistic).

Furthermore, the radiosensitizing effect of IUdR (IPdR) is independentof MGMT promoter methylation status, so it is useful across themolecular spectrum of high-grade gliomas.

Although not routinely analyzed, deficiencies in MMR and BER mechanismshave been documented in many high-grade gliomas, and tumors with thesedefects would be expected to derive even greater benefit from theaddition of IPdR to their RT regimen.

The patient will initially undergo maximal surgical resection, with agoal of <5% residual disease (the infiltrative nature of high-gradegliomas dictates this goal). Prior to the initiation of RT, IPdR will beadministered to the patient in the form of an oral drug (optimal dose ofIPdR to be determined in ongoing clinical trials, predicted to be in therange of 0.1-50 gm/M2/day, preferably 2 to 5 gm/M2/day). The patientwill begin TMZ therapy (75-150 mg/M2/day)+RT (typically, 60 Gy deliveredin 2 Gy fractions, 1 fraction/day, 5 days/week×6 weeks) one weekfollowing the initiation of IPdR. The high therapeutic index of IPdR(minimal toxicity encountered at clinically effective dose), andspecifically the lack of bone marrow suppression (i.e. will not augmentthe dose limiting toxicity of TMZ (bone marrow suppression)), allowsaddition of IPdR, with its radiation sensitizing effect, to the TMZ+RTregimen without the addition of significant toxicity. The patient willcontinue TMZ and IPdR throughout the course of RT. At the conclusion ofRT, IPdR will be discontinued, and the patient will proceed with TMZ ona new, maintenance dosage schedule (typically 150 mg/M2/day, days 1-5 ofa 28-day cycle, for 6 cycles).

The radiosensitizing effect of IPdR, in addition to the enhancement ofRT effect with TMZ in this MGMT promoter methylated GBM, will optimizetumor control/eradication for this patient, and prolongation of survival(e.g., by up to 70%, 24 months) is predicted.

Example 5

A 57 year old female has a 5 cm mass in the head of the pancreas withobstruction of the common bile duct and tumor encasing the superiormesenteric artery, rendering the tumor initially unresectable. Noevidence of metastatic disease is seen. This scenario occurs in up to35% of patients diagnosed with pancreatic cancer (approximately 28,000patients/year in the U.S.). Clinical trials in such patients withlocally advanced pancreas cancer have recently focused on combinationsof chemotherapy+RT to shrink the tumor enough that it becomesresectable. Such a tumor downstaging occurs in approximately 25% ofpatients.

Ribonucleotide reductase (RR) is an enzyme critical to a cell's recoveryfrom DNA damage caused by RT. RR is principally responsible formaintaining nucleotide pool balance and the DNA replication process thatis needed to repair RT-induced DNA damage.

Ribonucleotide reductase inhibitors (RRI) including hydroxyurea,gemcitabine, triapine and COH29, function as radiation sensitizers byaltering the nucleotide pool composition and inhibiting repair ofRT-induced DNA damage. The addition of IPdR to RRI administration causesfurther inhibition of DNA damage repair, in a synergistic (i.e. greaterthan additive) manner. Gemcitabine (2′,2′-difluoro-2′-deoxycitidine,dFdc) is a cytidine analog and an active chemotherapy drug in patientswith metastatic pancreas cancer. It is a prodrug that requires cellularuptake and intracellular phosphorylation to gemcitabine di- andtriphosphates (dFdCDP and dFdCTP, respectively), which are the activedrug metabolites. dFdCTP potentially inhibits RR and is believed to be amechanism for radiosensitization as previously described.

In this example, IPdR will be given in conjunction with gemcitabine+RTwith the goal of enhancing the degree of tumor reduction to make itamenable to surgical resection. Prior to the initiation of RT, IPdR willbe administered to the patient in the form of an oral drug (optimal doseof IPdR to be determined in ongoing clinical trials, predicted to be inthe range of 0.1-50 gm/M2/day, preferably 2 to 5 gm/M2/day). The patientwill begin gemcitabine therapy (typically, 1000 mg/M2/dose IV q week)1-4 weeks (preferably 1-2 weeks, typically 1 week) following theinitiation of IPdR, and undergo RT (typically, 54 Gy total dosedelivered in 1.8 Gy fractions, 1 fraction/day, 5 days per week). Thepatient will continue to take oral IPdR in addition to receivinggemcitabine therapy throughout the course of RT, and will discontinueIPdR following completion of RT. The high therapeutic index of IPdR(minimal toxicity encountered at clinically effective dose), andspecifically the lack of myelosuppression (the dose limiting toxicity ofgemcitabine) allows addition of IPdR, with its radiation sensitizingeffect, to the gemcitabine+RT regimen without the addition ofsignificant toxicity. Further inhibition, by IPdR, of repair ofRT-induced DNA damage resulting from RRI+RT therapy will optimize tumorcontrol/eradication.

The patient's CT scan post-IPdR+gemcitabine+RT shows that it istechnically resectable, and upon pathologic inspection, the margins arefree of tumor (i.e. a RO-resection), a scenario that occurs in up to 40%of patients who undergo resection following RT-induced downstaging, withthese patients' median survival approaches 2 years compared to less than1 year for patients who remain unresectable following RT. Given thepreclinical data combining IUdR (or IPdR)+RRI (e.g. gemcitabine)+RT, thedrug-drug and drug-drug-RT greater than additive interactions of thiscombination would be expected to further increase the percentage ofpatients that are able to achieve an RO-resection (e.g., from 40% to>60%).

Example 6

A 60 year old male non-smoker has a 6 cm distal esophagus mass. Biopsyreveals a squamous cell cancer, and immunohistochemical analysis of thespecimen reveals reduced expression of the base excision repair (BER)enzyme, MYH, and increased 8-oxoguanine oxidative damage in tumorcompared to normal esophagus, suggesting a BER-deficient cancerresulting from chronic gastric reflux injury. Clinically, BER-deficienttumors are aggressive, often with regional lymph node involvement.Evaluation demonstrates that this patient has Stage 3B disease, and thetreatment plan will include chemotherapy, RT and surgery, with anexpected 5-years survival rate of 20%. Complete surgical resection isimperative for cure, and so neoadjuvant therapy is administered with thegoal of downstaging the patient's disease and rendering the tumor moreamenable to resection.

In this example, IPdR will be given in conjunction withcis-platin+5-FU+RT with the goal of enhancing the degree of tumorreduction to allow surgical resection. Prior to the initiation of RT,IPdR will be administered to the patient in the form of an oral drug(optimal dose of IPdR to be determined in ongoing clinical trials,predicted to be in the range of 0.1-50 gm/M2/day, preferably 2 to 5gm/M2/day). The patient will begin cis-platin therapy (typically, 100mg/M2/dose IV on days 1 and 29) and continuous infusion 5-FU (1000mg/M2/day, days 1-4 and 29-32) 1-4 weeks (preferably 1-2 weeks,typically 1 week) following the initiation of IPdR, and undergo RT(typically, 50.4 Gy total dose delivered in 1.8 Gy fractions, 1fraction/day, 5 days per week). The patient will continue to take oralIPdR throughout the course of RT, and will discontinue IPdR followingcompletion of RT. The high therapeutic index of IPdR (minimal toxicityencountered at clinically effective dose), and specifically, the lack ofrenal toxicity, ototoxicity, peripheral neuropathy, myelosuppression andgastrointestinal toxicity (the dose limiting toxicities ofcis-platin+5-FU) allows addition of IPdR, with its radiation sensitizingeffect, to the cis-platin+5-FU+RT regimen without the addition ofsignificant toxicity.

The addition of IPdR to the standard regimen would exploit theBER-deficiency of the patient's tumor for therapeutic benefit. Based onthe preclinical data, the BER-deficiency would result in increasedIUdR-DNA incorporation in the tumor cells because they would be lessable to repair the G:IU adducts. This augmentation of response would bein addition to the known greater than additive drug-drug interactions,IUdR (IPdR)+cis-platin and IUdR (IPdR)+5-FU (via IdUMP binding(inhibiting) thymidylate synthase; as well as the IUdR (IPdR)+RTsynergism. This augmentation of response would be predicted to result ingreater tumor reduction, and thus increasing the likelihood of completesurgical resection, culminating in improved survival.

Example 7

A 38 year old female, non-smoker, presents with a persistent cough andchest x-ray reveals a 6 cm right upper lung mass. Further evaluation andbiopsy demonstrate a Stage 3B (T3 N3 MO) adenocarcinoma of the lung.Molecular testing of the tumor shows no mutations in the ALK or EGFRgenes, but does reveal promoter methylation of hMLH1, a MMR gene (i.e.MMR-deficient tumor). MMR deficiency is present in approximately 50% oflung cancers in never smokers, and is associated with a poor prognosis.

Standard-of-care for this patient is either cis-platin+etoposide+RT orRT alone. The poor response of MMR-deficient tumors compared to thosethat are MMR-proficient can be predicted on the basis of theMMR-deficiency because these tumors have been shown to be“damage-tolerant” (i.e. resistant) to the specific DNA damage caused bycytotoxic chemotherapy and ionizing radiation (IR). IUdR-mediatedradiosensitization results from the formation of G:IU DNA adducts that,once generated, are also tolerated by MMR-deficient tumors. Theadministration of po IPdR generates a 2-3-fold increase in IUdR-DNAincorporation compared to the administration of ci IUdR, enhancing thedegree of radiosensitization and overcoming the resistance mechanismsused by MMR-deficient tumors. Thus, the addition of IPdR to achemotherapy and/or RT regimen would be expected to augment tumorresponse in MMR-deficient tumors. In this example, prior to theinitiation of RT, IPdR will be administered to the patient in the formof an oral drug (optimal dose of IPdR to be determined in ongoingclinical trials, predicted to be in the range of 0.1-50 gm/M2/day,preferably 2 to 5 gm/M2/day). The patient will begin cis-platin therapy(typically, 50 mg/M2/dose IV on days 1, 8, 29 and 36) and etoposide (50mg/M2/day, days 1-5 and 29-33) 1-4 weeks (preferably 1-2 weeks,typically 1 week) following the initiation of IPdR, and undergo RT(typically, 60 Gy total dose delivered in 2 Gy fractions, 1fraction/day, 5 days per week). The patient will continue to take oralIPdR throughout the course of RT, and will discontinue IPdR followingcompletion of RT. The high therapeutic index of IPdR (minimal toxicityencountered at clinically effective dose), and specifically, the lack ofrenal toxicity, ototoxicity, peripheral neuropathy, myelosuppression andgastrointestinal toxicity (the dose limiting toxicities ofcis-platin+etoposide) allows addition of IPdR, with its radiationsensitizing effect, to the cis-platin+etoposide+RT regimen without theaddition of significant toxicity. The expectation would be improvedtumor control with this regimen compared to the identical regimenwithout the addition of IPdR. Furthermore, if, for any reason, a patientwith such an MMR-deficient tumor receives RT alone (as opposed tochemotherapy+RT), the addition of IPdR to the RT regimen would beexpected to produce similar, improved results.

Example 8

Hypofractionated RT using stereotactic radiosurgery technique. A 75 yearold female smoker is diagnosed with a 4 cm T2 N0 M0 squamous cellcarcinoma of the lung. Although surgical resection would provide thebest chance for cure, the patient has severe chronic obstructivepulmonary disease as a consequence of her smoking habits, and is not acandidate for surgery.

This patient will be treated with RT alone, specifically using astereotactic radiosurgery (SRS) technique, where the total dose of RT isadministered over a short timeframe (typically 1-14 days) in a limitednumber (typically 1-5) of doses, with each dose delivering a large IRfraction (typically, 8-20 Gy). Stereotactic techniques are appropriatefor small (typically, <5 cm) primary lung tumors or lung metastases awayfrom central structures (e.g. trachea, bronchus and heart). If RS isadministered at full dose (50-60 Gy), primary lung tumors demonstrate anup to 80% response rate without excessive normal tissue toxicity. Inthis example, the central location of the patient's tumor dictates thatparts of the esophagus, trachea, bronchus and heart will be in the SRSfield, thus limiting the dose to (40 Gy total dose, delivered as 8 Gyfractions, 5 fractions administered over 14 days), and thereby alsocompromising response rate, which under these circumstances is expectedto be ≦50%. The patient in this example will receive, prior to theinitiation of SRS, IPdR in the form of an oral drug (optimal dose ofIPdR to be determined in ongoing clinical trials, predicted to be in therange of 0.1-50 gm/M2/day, preferably 2 to 5 gm/M2/day). One-four weeks(preferably 1-2 weeks, typically 1 week) following the initiation ofIPdR, the patient will undergo SRS. The patient will continue to takeoral IPdR throughout the course of SRS, and will discontinue IPdRfollowing completion of SRS. The high therapeutic index of IPdR (minimaltoxicity encountered at clinically effective dose), and specifically,the lack of cardiac or mucosal toxicities, allows addition of IPdR, withits radiation sensitizing effect, to the SRS regimen without theaddition of significant toxicity. Based on IPdR's RT enhancement ratioof 1.3-6.0 as determined in preclinical studies, this patient's responserate would be predicted to improve.

The addition of IPdR to SRS regimens is especially valuable becausethese are situations in which RT is typically the only treatment aimedat local tumor control. Other examples of these situations include(lesions typically must be <5 cm) primary of metastatic liver tumors,and primary or metastatic brain and spinal cord tumors. Theradiosensitization effect (1.3-6.0×) of IPdR is predicted to enhance theeffectiveness of SRS techniques to a clinically significant degree.

Example 9

A 27 year-old female presents with a chordoma arising from the base ofthe skull (BOS). Chordomas are low-grade malignancies with lowmetastatic potential, and although the clinical course is slow (i.e.years), local progression and recurrence following resection occur inthe majority of patients. Surgical resection is critical, but almostalways limited by their critical location (i.e. arising from notochordremnants, chordomas inherently involve critical neurological structures)and infiltration into adjacent bone. To overcome the potential evolutionof residual disease, RT is used in the postoperative setting. Chordomasrespond best to high doses (in the range of 70 Gy) of radiation, butthese tumors represent a challenge for RT because nearby criticalneurologic structures (spinal cord, brainstem, and optic pathways) limitthe doses that can be delivered. Charged particles, namely protons, havebeen used in addition or instead of photons for their distinct advantageover conventional RT because of the superior dose distribution due tothe rapid radiation fall-off beyond the target.

For BOS chordomas, proton therapy (PT) is the adjuvant (i.e.post-surgical resection) treatment of choice, with doses in the range of60-95 Gy equivalent. The highest doses of PT are, predictably,associated with greater rates of tumor control, but at the expense oftoxicity to surrounding vital neurological structures. The mechanism ofcytotoxicity from PT is identical to that of conventional RT, i.e.IR-induced DNA damage, therefore, the radiosensitization effect of IPdRwould be predicted to apply when administered in conjunction with PT.

The patient will receive, prior to the initiation of PT, IPdR in theform of an oral drug (optimal dose of IPdR to be determined in ongoingclinical trials, predicted to be in the range of 0.1-50 gm/M2/day,preferably 2 to 5 gm/M2/day). One-four weeks (preferably 1-2 weeks,typically 1 week) following the initiation of IPdR, the patient willundergo PT (total dose 60-95 Gy-equivalent, 1.8-3.5 Gy-equivalentfractions, 1 fraction/day, 5 fractions/week). The patient will continueto take oral IPdR throughout the course of PT, and will discontinue IPdRfollowing completion of PT. The high therapeutic index of IPdR (minimaltoxicity encountered at clinically effective dose), and specifically,the lack of neurologic toxicity, allows addition of IPdR, with itsradiation sensitizing effect, to the PT regimen without the addition ofsignificant toxicity. Based on IPdR's RT enhancement ratio of 1.3-6.0 asdetermined in preclinical studies, local control and overall survival ispredicted to improve in this patient.

PT as a modality of RT is gaining popularity as it allows delivery ofhigher doses of IR with equivalent or less toxicity (i.e. the TI of PTis higher than that of conventional RT). The use of PT, however, islimited by availability (limited number of PT centers), feasibility(logistics involved in the administration of PT formidable), and thecost (cost of PT is, on average, 3-5 times the cost of conventional RT).The obstacles of PT justify its use in cases where higher RT dosesrepresent the best, and often only, chance for survival, and in thosecases, the addition of IPdR (with it's predicted radiosensitization) toPT is predicted to result in significant therapeutic benefit (i.e.increases the TI of the IR by up to 1.5×).

Example 10

Therapeutic Index (TI) is an important concept in considering theutility of a drug in clinical practice. In general, TI is a quantitativerelationship between the safety (toxicology) of a drug and its efficacy(pharmacology). A drug's TI is a ratio between two doses, the dose ofthe drug that causes adverse effects at an incidence/severity notcompatible with the targeted indication divided by the dose that leadsto the desired pharmacological effect, and the value of the TI isdependent upon the choice of doses used to calculate the ratio. TI is auseful concept for comparing different drugs used in the same situation,wherein identical requirements for efficacy and toxicity can be appliedto both drugs. TI is also useful as a semi-quantitative measure, whereina drug is demonstrated to have a low vs high TI, with higher TI beingdesirable.

In the case of IPdR (IUdR), it is appropriate to consider the TI interms of % IUdR-DNA cellular incorporation. Preclinical studies haveconfirmed and quantified the direct relationship between the dose ofIPdR administered and the % IUdR-DNA cellular incorporation, so the %incorporation may be used for TI calculations in place of the actualdose of IPdR:

${TI}_{IPdR} = \frac{{\% \mspace{14mu} {IUdR}} - {{DNA}\mspace{14mu} {cellular}\mspace{14mu} {incorporation}\mspace{14mu} {in}\mspace{14mu} {tumor}\mspace{14mu} {cells}}}{{\% \mspace{14mu} {IUdR}} - {{DNA}\mspace{14mu} {cellular}\mspace{14mu} {incorporation}\mspace{14mu} {in}\mspace{14mu} {normal}\mspace{14mu} {tissue}{\mspace{11mu} \;}{cells}}}$

Using this formula, the TI of ci IUdR in the clinical studies (Tables 1and 2) is calculated using the values (% IUdR-DNA incorporation) atwhich grade 3 or 4 (i.e. dose limiting) toxicities (myelosuppression,gastrointestinal and hepatic) were encountered, revealing a TIapproximately equal to 1:

${TI}_{ciIUdR} = {\frac{3 - {8\%}}{4 - {6\%}} \cong 1}$

Although the TI_(ci IUdR) in these studies was relatively low, ci IUdRat these doses did enhance tumor control in typically “radioresistant”(poorly RT-responsive) tumors, including glioblastoma multiforme (GBM)and high-grade sarcomas.

From the preclinical studies using human tumor xenografts, (MMR⁺colorectal cancers and GBM), the following TI_(ci IUdR) can becalculated:

${TI}_{ciIUdR} = {\frac{2 - {4\%}}{3 - {5\%}} \cong 1}$${TI}_{ciIUdR} = {{\frac{2 - {4\%}}{3 - {5\%}}x} \leq 1}$

(ci IUdR×6-14 days; GI toxicity of >20% body weight loss) and:

${TI}_{poIPdR} = {\frac{4 - {6\%}}{1 - {2\%}} \cong {2 - 6}}$

(po IPdR×14 days; no myelosuppression and <10% body weight loss).

Example 11

The observed TI_(po IPdR) seen in the initial Phase I and II clinicaltrials of po IPdR in Example 10 will be confirmed.

In the ongoing clinical trial of po IPdR in patients withgastrointestinal cancers (including patients with metastases), a tumorbiopsy will be obtained following 7-28 days of po IPdR (dose of IPdR tobe determined in ongoing clinical trials, predicted to be in the rangeof 0.1-50 gm/M2/day, preferably 2 to 5 gm/M²/day) (i.e. prior to theinitiation of RT, which is anticipated to begin 7-28 days following theinitiation of po IPdR). Tumor biopsies are obtained prior to RT toeliminate any cytotoxic effect of IR on the tumor in the analyses of %IUdR-DNA cellular incorporation. Samples of normal tissues includingcirculating granulocytes (as a surrogate of bone marrow) and oralmucosal scrapings (as a surrogate of gastrointestinal tract) will beobtained at the same time (i.e. prior to the initiation of RT) and alsoat weekly intervals throughout IPdR+RT. The cells obtained at thesetimes will be analyzed for % IUdR-DNA cellular incorporation, and thevalues used to calculate the TI.

Example 12

Attempt to further increase the TI of po IPdR by altering the “loading”schedule of po IPdR prior to the initiation of RT.

-   -   a. Administration of po IPdR TID×7 days before the initiation of        RT. Based on the plasma pharmacokinetics of po IPdR (i.e. peak        plasma levels of the IUdR (active drug)>4 μM×4 hours following a        po IPdR dose of 2400 mg, with IUdR levels decreasing to 1 μM        over 8 hours), a TID dosing schedule (as compared to once QD)        will more closely mimic the ci IUdR exposure (steady state        plasma concentration of 1 μM) that produced clinical responses        in the clinical studies of ci IUdR (Tables 1 and 2).        -   PO IPdR (dose of IPdR to be determined in ongoing clinical            trials, predicted to be in the range of 0.1-50 gm/M2/day            divided into 3 equal doses, preferably 2 to 5 gm/M²/day)            will be administered prior to the initiation of RT (7 days            following the initiation of po IPdR). A tumor biopsy will be            obtained immediately prior to the initiation of RT (i.e.            following 7 of po IPdR). Samples of normal tissues including            circulating granulocytes (as a surrogate of bone marrow) and            oral mucosal scrapings (as a surrogate of gastrointestinal            tract) will be obtained at the same time (i.e. prior to the            initiation of RT) and also at weekly intervals throughout            IPdR+RT. The cells obtained at these times will be analyzed            for % IUdR-DNA cellular incorporation, and the values used            to calculate the TI of the TID dosing administration            schedule of IPdR.    -   b. Administration of po IPdR QD×14-28 days before the initiation        of RT. Based on the plasma pharmacokinetics of po IPdR (i.e.        peak plasma levels of the IUdR (active drug)>4 μM×4 hours        following a po IPdR dose of 2400 mg, with IUdR levels decreasing        to 1 μM over 8 hours), a prolonged exposure (i.e. 14-28 days        compared to 7 days) to po IPdR will more closely mimic the ci        IUdR exposure that produced clinical responses in the clinical        studies of ci IUdR (Tables 1 and 2).        -   PO IPdR (dose of IPdR to be determined in ongoing clinical            trials, predicted to be in the range of 0.1-50 gm/M2/day,            preferably 2 to 5 gm/M²/day) will be administered prior to            the initiation of RT, to begin 14-28 days following the            initiation of po IPdR. A tumor biopsy will be obtained            immediately prior to the initiation of RT (i.e. following            14-28 days of po IPdR). Samples of normal tissues including            circulating granulocytes (as a surrogate of bone marrow) and            oral mucosal scrapings (as a surrogate of gastrointestinal            tract) will be obtained at the same time (i.e. prior to the            initiation of RT) and also at weekly intervals throughout            IPdR+RT. The cells obtained at these times will be analyzed            for % IUdR-DNA cellular incorporation, and the values used            to calculate the TI of the TID dosing administration            schedule of IPdR.

Comparison will then be made between the calculated TIs, using %IUdR-DNA cellular incorporation levels, for po IPdR administered via thethree different dosing (i.e. “loading”) schemes:

QD dosing for 7 days vs

QD dosing for 14-28 days vs

TID dosing for 7 days

prior to the initiation of RT.

Example 13

In conjunction with the calculation and comparison of the TIs of po IPdRadministered via three different dosing schedules in Example 12 above,for each of the dosing regimes, prediction of the sensitizer enhancementratio (SER) in tumor will be made by measuring the proportion ofIUdR-DNA labeled (vs unlabeled) tumor cells by flow cytometry.

The degree of IPdR-mediated radiosensitization is directly related tothe % IUdR-DNA cellular incorporation, and because intracellular IdUTPpools are available for IUdR-DNA incorporation only during the S-phase(DNA synthesis phase) of the cell cycle, the number of unlabeled cellsmust be minimized for maximal radiosensitization. Experimental andmathematical work has derived and validated the following formula tocalculate the SER:

${{Tumor}\mspace{14mu} {SER}} = {1 + {\frac{- {\log_{10}\left( {{proportion}\mspace{14mu} {of}\mspace{14mu} {unlabeled}\mspace{14mu} {cells}} \right)}}{\log_{10}\left( {{total}\mspace{14mu} {number}\mspace{14mu} {of}\mspace{14mu} {viable}\mspace{14mu} {cells}} \right)}.}}$

Using the previously described flow cytometry techniques with anti-IUdRantibodies, analyses of single cell aliquots of the tumor biopsyspecimens (obtained immediately prior to the initiation of RT, following7-28 days of po IPdR) will be used for the calculations of Tumor SERs.

The aspects, embodiments, features, and examples of the invention are tobe considered illustrative in all respects and are not intended to limitthe invention, the scope of which is defined only by the claims. Otherembodiments, modifications, and usages will be apparent to those skilledin the art without departing from the spirit and scope of the claimedinvention.

The use of headings and sections in the application is not meant tolimit the invention; each section can apply to any aspect, embodiment,or feature of the invention.

Throughout the application, where compositions are described as having,including, or comprising specific components, or where processes aredescribed as having, including or comprising specific process steps, itis contemplated that compositions of the present teachings also consistessentially of, or consist of, the recited components, and that theprocesses of the present teachings also consist essentially of, orconsist of, the recited process steps.

In the application, where an element or component is said to be includedin and/or selected from a list of recited elements or components, itshould be understood that the element or component can be any one of therecited elements or components and can be selected from a groupconsisting of two or more of the recited elements or components.Further, it should be understood that elements and/or features of acomposition, an apparatus, or a method described herein can be combinedin a variety of ways without departing from the spirit and scope of thepresent teachings, whether explicit or implicit herein.

The use of the terms “include,” “includes,” “including,” “have,” “has,”or “having” should be generally understood as open-ended andnon-limiting unless specifically stated otherwise.

It should be understood that the order of steps or order for performingcertain actions is immaterial so long as the present teachings remainoperable. Moreover, two or more steps or actions may be conductedsimultaneously.

The use of the singular herein includes the plural (and vice versa)unless specifically stated otherwise. Moreover, the singular forms “a,”“an,” and “the” include plural forms unless the context clearly dictatesotherwise. In addition, where the use of the term “about” is before aquantitative value, the present teachings also include the specificquantitative value itself, unless specifically stated otherwise. As usedherein, the term “about” refers to a ±10% variation from the nominalvalue.

Where a range or list of values is provided, each intervening valuebetween the upper and lower limits of that range or list of values isindividually contemplated and is encompassed within the invention as ifeach value were specifically enumerated herein. In addition, smallerranges between and including the upper and lower limits of a given rangeare contemplated and encompassed within the invention. The listing ofexemplary values or ranges is not a disclaimer of other values or rangesbetween and including the upper and lower limits of a given range.

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting on the invention described herein. Scope of theinvention is thus indicated by the appended claims rather than by theforegoing description, and all changes which come within the meaning andrange of equivalency of the claims are intended to be embraced therein.

TABLE 1 NCI sponsored clinical efficacy studies of IUdR (compared tocontemporary historical RT-alone controls) for treatment of high-gradeprimary brain tumors (RTOG*, NCI** trials) Median survival TumorTreatment (Months) Anaplastic astrocytomas (Grade 3 of 4)* RT alone 24RT + IUdR 39 Glioblastoma Multiforme (Grade 4 of 4)** RT alone 9 RT +IUdR 15

TABLE 2 Clinical efficacy studies of IUdR (compared to historicalRT-alone controls) for treatment of high-grade sarcomas (NCI**;University of Michigan*** trials) Local Control at Tumor Treatment 2years High grade sarcomas RT + Surgery 25% (resectable)*** RT + IUdR +Surgery 45% High grade sarcomas RT alone <10%  (un-resectable)** RT +IUdR 60%

TABLE 3 Summary of IPdR pre-clinical studies leading to IND applicationand initial clinical Phase 0 trial Study Description Summary of FindingsA. IPdR Metabolism by hepatic aldehyde oxidase Chang, 1992 Metabolism ofElucidated IPdR metabolism IPdR vs IUdR by aldehyde oxidaseCharacterized properties of aldehyde oxidase Kinsella, 1998, IPdRMetabolism Characterized kinetics of 2000, 2008 in rats and ferrets IPdRmetabolism and in human tissues B. Pharmacokinetic and toxicologystudies Kinsella, 1994, Pharmacokinetics of Established absorption,1998, 2000 po IPdR in mice distribution, metabolism, and eliminationkinetics of po IPdR in mice Kinsella, 2000 Pharmacokinetics (PK)Established distribution, and toxicity/toxicology metabolism, andelimination of IPdR in ferrets (po) kinetics of IPdR in non- and rhesusmonkeys (iv) rodent species. Noted mild weight loss at highest dose; butno significant hematologic, biochemical, or histopathologic changesKinsella, 2008 Pharmacokinetics Established IPdR and IUdR and toxicity/concentration-time profiles toxicology Reported HPLC/tandem of po IPdRin mass spectroscopy methods Fischer rats for plasma IPdR and IUdRlevels. Kummar, 2013 Pharmacokinetics Phase 0 study of po IPdR, ofsingle-dose 150 mg-2400 mg in humans: po IPdR in humans No toxicities.C. Pre-clinical efficacy studies of IPdR-mediated radiosensitizationKinsella, 1998, Efficacy/toxicity Increased IUdR-DNA 2000 studies of poincorporation in tumors; Seo, 2004, IPdR vs ci decreased in normaltissues, 2005 IUdR using human po IPdR vs ci IUdR colorectal and 1.3-1.5fold enhancement of glioblastoma response to RT with po IPdR tumorxenografts. Kinsella, 1994 Efficacy, PK, Demonstrated improved toxicity,and DNA therapeutic index of po IPdR incorporation vs po IUdR for IUdR-of po IPdR vs po mediated radiosensitization IUdR in human colon cancerxenografts.

What is claimed is:
 1. A method of treating a human patient having cancer, the method comprising the steps of: administering IPdR to the patient in the form of an oral drug; and administering radiation therapy (RT) to the patient.
 2. The method of claim 1, wherein RT is administered via a hyperfractionated technique.
 3. The method of claim 1, wherein the patient cannot tolerate the optimum RT field without the addition of IPdR.
 4. The method of claim 1, wherein the technique of RT delivery is selected from the group of: 3-dimensional conformal radiation therapy, intensity-modulated radiation therapy, image-guided radiation therapy, tomotherapy, stereotactic radiosurgery and stereotactic body radiation therapy.
 5. The method of claim 1, wherein the source of RT is selected from the group consisting of: protons and carbon ions.
 6. The method of claim 1, wherein the cancer is MMR-deficient.
 7. The method of claim 1 further comprising the step of: administering a chemotherapeutic drug or biologic agent to the patient prior to administering radiation therapy (RT) to the patient.
 8. The method of claim 7, wherein the chemotherapeutic drug is selected from the group consisting of a fluoropyrimidine, a platinum analog, a ribonucleotide reductase inhibitor and a methylating agent.
 9. The method of claim 7, wherein the biologic agent is a BER modulator.
 10. The method of claim 7, wherein the cancer is BER-deficient.
 11. The method of claim 1, wherein the IPdR is administered at a dose of 0.1-50 gm/M2/day.
 12. A method of optimizing IPdR sensitization for radiation therapy for a cancer patient having been administered IPdR, the method comprising: determining an IUdR-DNA incorporation level in a first tumor biopsy taken from the patient; determining an IUdR-DNA incorporation level in a normal tissue sample; and calculating a therapeutic index; wherein the therapeutic index guides dose and schedule of radiation therapy.
 13. The method of claim 12, wherein the therapeutic index is calculated by dividing the percent IUdR-DNA incorporation level in the first tumor biopsy by the percent IUdR-DNA incorporation of the normal tissue sample.
 14. The method of claim 12, wherein IUdR-DNA incorporation levels are measured by high performance liquid chromatography (HPLC) or flow cytometry.
 15. The method of claim 12, comprising determining an IUdR-DNA incorporation level in a second tumor biopsy taken at a second time from the patient, determining an IUdR-DNA incorporation level in a normal tissue sample; and calculating a therapeutic index. 